Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded tumour antigen

ABSTRACT

The present invention relates to a nucleic acid sequence, comprising or coding for a coding region, encoding at least one peptide or protein comprising a tumour antigen or a fragment, variant or derivative thereof, at least one histone stem-loop and a poly(A) sequence or a polyadenylation signal. Furthermore the present invention provides the use of the nucleic acid for increasing the expression of said encoded peptide or protein. It also discloses its use for the preparation of a pharmaceutical composition, especially a vaccine, e.g. for use in the treatment of cancer or tumour diseases. The present invention further describes a method for increasing the expression of a peptide or protein comprising a tumour antigen or a fragment, variant or derivative thereof, using the nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal.

This application is a continuation of U.S. application Ser. No.14/378,572, filed Aug. 13, 2014, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/EP2013/000459, filed Feb. 15, 2013, which is a continuation ofInternational Application No. PCT/EP2012/000674, filed Feb. 15, 2012.The entire text of each of the above referenced disclosures isspecifically incorporated herein by reference.

The present invention relates to a nucleic acid sequence, comprising orcoding for a coding region, encoding at least one peptide or proteincomprising a tumour antigen or a fragment, variant or derivativethereof, at least one histone stem-loop and a poly(A) sequence or apolyadenylation signal. Furthermore the present invention provides theuse of the nucleic acid for increasing the expression of said encodedpeptide or protein. It also discloses its use for the preparation of apharmaceutical composition, especially a vaccine, e.g. for use in thetreatment of cancer or tumour diseases. The present invention furtherdescribes a method for increasing the expression of a peptide or proteincomprising a tumour antigen or a fragment, variant or derivativethereof, using the nucleic acid comprising or coding for a histonestem-loop and a poly(A) sequence or a polyadenylation signal.

Apart from cardiovascular diseases and infectious diseases, theoccurrence of tumours and cancer diseases is one of the most frequentcauses of death in modern society and is in most cases associated withconsiderable costs in terms of therapy and subsequent rehabilitationmeasures. The treatment of tumours and cancer diseases is greatlydependent, for example, on the type of tumour that occurs, on the age,the distribution of cancer cells in the patient to be treated, etc.Cancer therapy is nowadays conventionally carried out by the use ofradiation therapy or chemotherapy in addition to invasive operations.However, such conventional therapies typically place extraordinarystress on the immune system and can be used in some cases to only alimited extent. In addition, most of these conventional therapiesrequire long intervals between the individual treatments to allow forregeneration of the immune system.

Therefore, supplementary strategies have been investigated in recentyears in addition to such “conventional treatments” to avoid or at leastreduce the impact on the immune system by such treatments. One suchsupplementary treatment in particular includes gene therapeuticapproaches or genetic vaccination, which already have been found to behighly promising for treatment or for supporting such conventionaltherapies.

Gene therapy and genetic vaccination are methods of molecular medicinewhich already have been proven in the therapy and prevention of diseasesand generally exhibit a considerable effect on daily medical practice,in particular on the treatment of diseases as mentioned above. Bothmethods, gene therapy and genetic vaccination, are based on theintroduction of nucleic acids into the patient's cells or tissue andsubsequent processing of the information coded for by the nucleic acidthat has been introduced into the cells or tissue, that is to say the(protein) expression of the desired polypeptides.

In gene therapy approaches, typically DNA is used even though RNA isalso known in recent developments. Importantly, in all these genetherapy approaches mRNA functions as messenger for the sequenceinformation of the encoded protein, irrespectively if DNA, viral RNA ormRNA is used.

In general RNA is considered an unstable molecule: RNases are ubiquitousand notoriously difficult to inactivate. Furthermore, RNA is alsochemically more labile than DNA. Thus, it is perhaps surprising that the“default state” of an mRNA in a eukaryotic cell is characterized by arelative stability and specific signals are required to accelerate thedecay of individual mRNAs. The main reason for this finding appears tobe that mRNA decay within cells is catalyzed almost exclusively byexonucleases. However, the ends of eukaryotic mRNAs are protectedagainst these enzymes by specific terminal structures and theirassociated proteins: a m7GpppN CAP at the 5′ end and typically a poly(A)sequence at the 3′ end. Removal of these two terminal modifications isthus considered rate limiting for mRNA decay. Although a stabilizingelement has been characterized in the 3′ UTR of the alpha-globin mRNA,RNA sequences affecting turnover of eukaryotic mRNAs typically act as apromoter of decay usually by accelerating deadenylation (reviewed inMeyer, S., C. Temme, et al. (2004), Crit Rev Biochem Mol Biol 39(4):197-216.).

As mentioned above, the 5′ ends of eukaryotic mRNAs are typicallymodified posttranscriptionally to carry a methylated CAP structure, e.g.m7GpppN. Aside from roles in RNA splicing, stabilization, and transport,the CAP structure significantly enhances the recruitment of the 40Sribosomal subunit to the 5′ end of the mRNA during translationinitiation. The latter function requires recognition of the CAPstructure by the eukaryotic initiation factor complex eIF4F. The poly(A)sequence additionally stimulates translation via increased 40S subunitrecruitment to mRNAs, an effect that requires the intervention ofpoly(A) binding protein (PABP). PABP, in turn, was recently demonstratedto interact physically with eIF4G, which is part of the CAP-bound eIF4Fcomplex. Thus, a closed loop model of translation initiation on capped,polyadenylated mRNAs was postulated (Michel, Y. M., D. Poncet, et al.(2000), J Biol Chem 275(41): 32268-76.).

Nearly all eukaryotic mRNAs end with such a poly(A) sequence that isadded to their 3′ end by the ubiquitous cleavage/polyadenylationmachinery. The presence of a poly(A) sequence at the 3′ end is one ofthe most recognizable features of eukaryotic mRNAs. After cleavage, mostpre-mRNAs, with the exception of replication-dependent histonetranscripts, acquire a polyadenylated tail. In this context, 3′ endprocessing is a nuclear co-transcriptional process that promotestransport of mRNAs from the nucleus to the cytoplasm and affects thestability and the translation of mRNAs. Formation of this 3′ end occursin a two step reaction directed by the cleavage/polyadenylationmachinery and depends on the presence of two sequence elements in mRNAprecursors (pre-mRNAs); a highly conserved hexanucleotide AAUAAA(polyadenylation signal) and a downstream G/U-rich sequence. In a firststep, pre-mRNAs are cleaved between these two elements. In a second steptightly coupled to the first step the newly formed 3′ end is extended byaddition of a poly(A) sequence consisting of 200-250 adenylates whichaffects subsequently all aspects of mRNA metabolism, including mRNAexport, stability and translation (Dominski, Z. and W. F. Marzluff(2007), Gene 396(2): 373-90.).

The only known exception to this rule are the replication-dependenthistone mRNAs which end with a histone stem-loop instead of a poly(A)sequence. Exemplary histone stem-loop sequences are described in Lopezet al. (Dávila López, M., & Samuelsson, T. (2008), RNA (New York, N.Y.),14(1), 1-10. doi: 10.1261/rna.782308.).

The stem-loops in histone pre-mRNAs are typically followed by apurine-rich sequence known as the histone downstream element (HDE).These pre-mRNAs are processed in the nucleus by a single endonucleolyticcleavage approximately 5 nucleotides downstream of the stem-loop,catalyzed by the U7 snRNP through base pairing of the U7 snRNA with theHDE. The 3′-UTR sequence comprising the histone stem-loop structure andthe histone downstream element (HDE) (binding site of the U7 snRNP) wereusually termed as histone 3′-processing signal (see e.g. Chodchoy, N.,N. B. Pandey, et al. (1991). Mol Cell Biol 11(1): 497-509.).

Due to the requirement to package newly synthesized DNA into chromatin,histone synthesis is regulated in concert with the cell cycle. Increasedsynthesis of histone proteins during S phase is achieved bytranscriptional activation of histone genes as well asposttranscriptional regulation of histone mRNA levels. It could be shownthat the histone stem-loop is essential for all posttranscriptionalsteps of histone expression regulation. It is necessary for efficientprocessing, export of the mRNA into the cytoplasm, loading ontopolyribosomes, and regulation of mRNA stability.

In the above context, a 32 kDa protein was identified, which isassociated with the histone stem-loop at the 3′-end of the histonemessages in both the nucleus and the cytoplasm. The expression level ofthis stem-loop binding protein (SLBP) is cell-cycle regulated and ishighest during S-phase when histone mRNA levels are increased. SLBP isnecessary for efficient 3′-end processing of histone pre-mRNA by the U7snRNP. After completion of processing, SLBP remains associated with thestem-loop at the end of mature histone mRNAs and stimulates theirtranslation into histone proteins in the cytoplasm. (Dominski, Z. and W.F. Marzluff (2007), Gene 396(2): 373-90). Interestingly, the RNA bindingdomain of SLBP is conserved throughout metazoa and protozoa (DávilaLópez, M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308) and it could be shown that its binding to thehistone stem-loop sequence is dependent on the stem-loop structure andthat the minimum binding site contains at least 3 nucleotides 5′ and 2nucleotides 3′ of the stem-loop (Pandey, N. B., et al. (1994), Molecularand Cellular Biology, 14(3), 1709-1720 and Williams, A. S., & Marzluff,W. F., (1995), Nucleic Acids Research, 23(4), 654-662.).

Even though histone genes are generally classified as either“replication-dependent”, giving rise to mRNA ending in a histonestem-loop, or “replacement-type”, giving rise to mRNA bearing apoly(A)-tail instead, naturally occurring mRNAs containing both ahistone stem-loop and poly(A) or oligo(A) 3′ thereof have beenidentified in some very rare cases. Sanchez et al. examined the effectof naturally occurring oligo(A) tails appended 3′ of the histonestem-loop of histone mRNA during Xenopus oogenesis using Luciferase as areporter protein and found that the oligo(A) tail is an active part ofthe translation repression mechanism that silences histone mRNA duringoogenesis and its removal is part of the mechanism that activatestranslation of histone mRNAs (Sanchez, R. and W. F. Marzluff (2004), MolCell Biol 24(6): 2513-25).

Furthermore, the requirements for regulation of replication dependenthistones at the level of pre-mRNA processing and mRNA stability havebeen investigated using artificial constructs coding for the markerprotein alpha Globin, taking advantage of the fact that the globin genecontains introns as opposed to the intron-less histone genes. For thispurpose constructs were generated in which the alpha globin codingsequence was followed by a histone stem-loop signal (histone stem-loopfollowed by the histone downstream element) and a polyadenylation signal(Whitelaw, E., et al. (1986). Nucleic Acids Research, 14(17),7059-7070.; Pandey, N. B., & Marzluff, W. F. (1987). Molecular andCellular Biology, 7(12), 4557-4559.; Pandey, N. B., et al. (1990).Nucleic Acids Research, 18(11), 3161-3170).

In another approach Lischer et al. investigated the cell-cycle dependentregulation of a recombinant histone H4 gene. Constructs were generatedin which the H4 coding sequence was followed by a histone stem-loopsignal and a polyadenylation signal, the two processing signalsincidentally separated by a galactokinase coding sequence (Lischer, B.et al., (1985). Proc. Natl. Acad. Sci. USA, 82(13), 4389-4393).

Additionally, Stauber et al. identified the minimal sequence required toconfer cell-cycle regulation on histone H4 mRNA levels. For theseinvestigations constructs were used, comprising a coding sequence forthe selection marker Xanthine:guanine phosphoribosyl transferase (GPT)preceding a histone stem-loop signal followed by a polyadenylationsignal (Stauber, C. et al., (1986). EMBO J, 5(12), 3297-3303).

Examining histone pre-mRNA processing Wagner et al. identified factorsrequired for cleavage of histone pre-mRNAs using a reporter constructplacing EGFP between a histone stem-loop signal and a polyadenylationsignal, such that EGFP was expressed only in case histone pre-mRNAprocessing was disrupted (Wagner, E. J. et al., (2007). Mol Cell 28(4),692-9).

To be noted, translation of polyadenylated mRNA usually requires the 3′poly(A) sequence to be brought into proximity of the 5′ CAP. This ismediated through protein-protein interaction between the poly(A) bindingprotein and eukaryotic initiation factor eIF4G. With respect toreplication-dependent histone mRNAs, an analogous mechanism has beenuncovered. In this context, Gallie et al. show that the histonestem-loop is functionally similar to a poly(A) sequence in that itenhances translational efficiency and is co-dependent on a 5′-CAP inorder to establish an efficient level of translation. They showed thatthe histone stem-loop is sufficient and necessary to increase thetranslation of a reporter mRNA in transfected Chinese hamster ovarycells but must be positioned at the 3′-terminus in order to functionoptimally. Therefore, similar to the poly(A) tail on other mRNAs, the 3′end of these histone mRNAs appears to be essential for translation invivo and is functionally analogous to a poly(A) tail (Gallie, D. R.,Lewis, N. J., & Marzluff, W. F. (1996), Nucleic Acids Research, 24(10),1954-1962).

Additionally, it could be shown that SLBP is bound to the cytoplasmichistone mRNA and is required for its translation. Even though SLBP doesnot interact directly with eIF4G, the domain required for translation ofhistone mRNA interacts with the recently identified protein SLIP1. In afurther step, SLIP1 interacts with eIF4G and allows to circularizehistone mRNA and to support efficient translation of histone mRNA by amechanism similar to the translation of polyadenylated mRNAs.

As mentioned above, gene therapy approaches normally use DNA to transferthe coding information into the cell which is then transcribed intomRNA, carrying the naturally occurring elements of an mRNA, particularlythe 5′-CAP structure and the 3′ poly(A) sequence to ensure expression ofthe encoded therapeutic or antigenic protein.

However, in many cases expression systems based on the introduction ofsuch nucleic acids into the patient's cells or tissue and the subsequentexpression of the desired polypeptides coded for by these nucleic acidsdo not exhibit the desired, or even the required, level of expressionwhich may allow for an efficient therapy, irrespective as to whether DNAor RNA is used.

In the prior art, different attempts have hitherto been made to increasethe yield of the expression of an encoded protein, in particular by useof improved expression systems, both in vitro and/or in vivo. Methodsfor increasing expression described generally in the prior art areconventionally based on the use of expression vectors or cassettescontaining specific promoters and corresponding regulation elements. Asthese expression vectors or cassettes are typically limited toparticular cell systems, these expression systems have to be adapted foruse in different cell systems. Such adapted expression vectors orcassettes are then usually transfected into the cells and typicallytreated in dependence of the specific cell line. Therefore, preferenceis given primarily to those nucleic acid molecules which are able toexpress the encoded proteins in a target cell by systems inherent in thecell, independent of promoters and regulation elements which arespecific for particular cell types. In this context, there can bedistinguished between mRNA stabilizing elements and elements whichincrease translation efficiency of the mRNA.

mRNAs which are optimized in their coding sequence and which are ingeneral suitable for such a purpose are described in application WO02/098443 (CureVac GmbH). For example, WO 02/098443 describes mRNAs thatare stabilised in general form and optimised for translation in theircoding regions. WO 02/098443 further discloses a method for determiningsequence modifications. WO 02/098443 additionally describespossibilities for substituting adenine and uracil nucleotides in mRNAsequences in order to increase the guanine/cytosine (G/C) content of thesequences. According to WO 02/098443, such substitutions and adaptationsfor increasing the G/C content can be used for gene therapeuticapplications but also genetic vaccines in the treatment of cancer orinfectious diseases. In this context, WO 02/098443 generally mentionssequences as a base sequence for such modifications, in which themodified mRNA codes for at least one biologically active peptide orpolypeptide, which is translated in the patient to be treated, forexample, either not at all or inadequately or with faults.Alternatively, WO 02/098443 proposes mRNAs coding for antigens e.g.tumour antigens or viral antigens as a base sequence for suchmodifications.

In a further approach to increase the expression of an encoded proteinthe application WO 2007/036366 describes the positive effect of longpoly(A) sequences (particularly longer than 120 bp) and the combinationof at least two 3′ untranslated regions of the beta globin gene on mRNAstability and translational activity.

However, even though all these latter prior art documents already try toprovide quite efficient tools for gene therapy approaches andadditionally improved mRNA stability and translational activity, therestill remains the problem of a generally lower stability of RNA-basedapplications versus DNA vaccines and DNA based gene therapeuticapproaches. Accordingly, there still exists a need in the art to provideimproved tools for gene therapy approaches and genetic vaccination or asa supplementary therapy for conventional treatments as discussed above,which allow for better provision of encoded proteins in vivo, e.g. via afurther improved mRNA stability and/or translational activity,preferably for gene therapy and genetic vaccination.

Furthermore despite of all progress in the art, efficient expression ofan encoded peptide or protein in cell-free systems, cells or organisms(recombinant expression) is still a challenging problem.

The object underlying the present invention is, therefore, to provideadditional and/or alternative methods to increase expression of anencoded protein, preferably via further stabilization of the mRNA and/oran increase of the translational efficiency of such an mRNA with respectto such nucleic acids known from the prior art for the use in geneticvaccination in the therapeutic or prophylactic treatment of cancer ortumour diseases.

This object is solved by the subject matter of the attached claims.Particularly, the object underlying the present invention is solvedaccording to a first aspect by an inventive nucleic acid sequencecomprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein        which comprises a tumour antigen or a fragment, variant or        derivative thereof;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal,        preferably for increasing the expression of said encoded peptide        or protein.

Alternatively, any appropriate stem loop sequence other than a histonestem loop sequence (derived from histone genes, in particular histonegenes of the families H1, H2A, H2B, H3 and H4)) may be employed by thepresent invention in all of its aspects and embodiments.

In this context it is particularly preferred that the inventive nucleicacid according to the first aspect of the present invention is producedat least partially by DNA or RNA synthesis, preferably as describedherein or is an isolated nucleic acid.

The present invention is based on the surprising finding of the presentinventors, that the combination of a poly(A) sequence or polyadenylationsignal and at least one histone stem-loop, even though both representingalternative mechanisms in nature, acts synergistically as thiscombination increases the protein expression manifold above the levelobserved with either of the individual elements. The synergistic effectof the combination of poly(A) and at least one histone stem-loop is seenirrespective of the order of poly(A) and histone stem-loop andirrespective of the length of the poly(A) sequence.

Therefore it is particularly preferred that the inventive nucleic acidsequence comprises or codes for a) a coding region, encoding at leastone peptide or protein which comprises a tumour antigen or a fragment,variant or derivative thereof; b) at least one histone stem-loop, and c)a poly(A) sequence or polyadenylation sequence; preferably forincreasing the expression level of said encoded peptide or protein. Insome embodiments, it may be preferred if the encoded protein is not ahistone protein, in particular no histone protein of the H4, H3, H2Aand/or H2B histone family or a fragment, derivative or variant thereofretaining histone(-like) function), namely forming a nucleosome. Also,the encoded protein typically does not correspond to a histone linkerprotein of the H1 histone family. The inventive nucleic acid moleculedoes typically not contain any regulatory signals (5′ and/or,particularly, 3′ of a mouse histone gene, in particular not of a mousehistone gene H2A and, further, most preferably not of the mouse histonegene H2A614. In particular, it does not contain a histone stem loopand/or a histone stem loop processing signal from a mouse histone gene,in particular not of mouse histone gene H2A und, most preferably not ofmouse histone gene H2A614.

Also, the inventive nucleic acid typically does not provide a reporterprotein (e.g. Luciferase, GFP, EGFP, β-Galactosidase, particularly EGFP)galactokinase (galK) and/or marker or selection protein (e.g.alpha-Globin, galactokinase and Xanthine:Guanine phosphoribosyltransferase (GPT)) or a bacterial reporter protein, e.g. chloramphenicolacetyl transferase (CAT) or other bacterial antibiotics resistanceproteins, e.g. derived from the bacterial neo gene in its element (a).

A reporter, marker or selection protein is typically understood not tobe a tumour antigen according to the invention. A reporter, marker orselection protein or its underlying gene is commonly used as a researchtool in bacteria, cell culture, animals or plants. They confer onorganisms (preferably heterologously) expressing them an easilyidentifiable property, which may be measured or which allows forselection. Specifically, marker or selection proteins exhibit aselectable function. Typically, such selection, marker or reporterproteins do not naturally occur in humans or other mammals, but arederived from other organisms, in particular from bacteria or plants.Accordingly, proteins with selection, marker or reporter functionoriginating from species other than mammals, in particular other thanhumans, are preferably excluded from being understood as a proteinhaving the property to act as a “tumour antigen” according to thepresent invention. In particular, a selection, marker or reporterprotein allows to identify transformed cells by in vitro assays basede.g. on fluorescence or other spectroscopic techniques and resistancetowards antibiotics. Selection, reporter or marker genes awarding suchproperties to transformed cells are therefore typically not understoodto be a protein acting as a tumour antigen according to the invention invivo.

In any case, reporter, marker or selection proteins do usually not exertany tumour antigenic properties and, therefore, do not exert animmunological effect which therapeutically allows to treat tumourdiseases. If any single reporter, marker or selection protein shouldnevertheless do so (in addition to its reporter, selection or markerfunction), such a reporter, marker or selection protein is preferablynot understood to be a “tumour antigen” within the meaning of thepresent invention.

In contrast, a protein or peptide acting as a tumour antigen (inparticular excluding histone genes of the families H1, H2A, H2B, H3 andH4) according to the present invention does typically not exhibit aselection, marker or reporter function. If any single “tumour antigen”nevertheless should do so (in addition to its tumour antigenicfunction), such a tumour antigen is preferably not understood to be a“selection, marker or reporter protein” within the meaning of thepresent invention.

It is most preferably understood that a protein acting as tumour antigenaccording to the invention is derived from mammals, in particularhumans, in particular from mammalian tumours, and does not qualify asselection, marker or reporter protein. In particular, such tumourantigens are derived from mammalian, in particular from human tumours.These tumour antigenic proteins are understood to be antigenic, as theyare meant to treat the subject by triggering the subject's immuneresponse such that the subject's immune system is enabled to combat thesubject's tumor cells by TH1 and/or TH2 immune responses. Accordingly,such antigenic tumour proteins are typically mammalian, in particularhuman proteins characterizing the subject's cancer type.

Accordingly, it is preferred that the coding region (a) encoding atleast one peptide or protein is heterologous to at least (b) the atleast one histone stem loop, or more broadly, to any appropriate stemloop. In other words, “heterologous” in the context of the presentinvention means that the at least one stem loop sequence does notnaturally occur as a (regulatory) sequence (e.g. at the 3′UTR) of thespecific gene, which encodes the (tumour antigenic) protein or peptideof element (a) of the inventive nucleic acid. Accordingly, the (histone)stem loop of the inventive nucleic acid is derived preferably from the3′ UTR of a gene other than the one comprising the coding region ofelement (a) of the inventive nucleic acid. E.g., the coding region ofelement (a) will not encode a histone protein or a fragment, variant orderivative thereof (retaining the function of a histone protein), if theinventive nucleic is heterologous, but will encode any other peptide orsequence (of the same or another species) which exerts a biologicalfunction, preferably tumour antigenic function other than ahistone(-like) function, e.g. will encode a protein (exerting a tumourantigenic function, e.g. in terms of vaccinating against mammalian, inparticular human tumours thereby triggering a immunological reactionagainst the subject's tumour cells, which preferably express the tumourantigen encoded by the inventive nucleic acid.

In this context it is particularly preferred that the inventive nucleicacid comprises or codes for in 5′- to 3′-direction:

-   -   a) a coding region, encoding at least one peptide or protein        which comprises a tumour antigen or a fragment, variant or        derivative thereof;    -   b) at least one histone stem-loop, optionally without a histone        downstream element (HDE) 3′ to the histone stem-loop    -   c) a poly(A) sequence or a polyadenylation signal.

The term “histone downstream element (HDE) refers to a purine-richpolynucleotide stretch of about 15 to 20 nucleotides 3′ of naturallyoccurring histone stem-loops, which represents the binding site for theU7 snRNA involved in processing of histone pre-mRNA into mature histonemRNA. For example in sea urchins the HDE is CAAGAAAGA (Dominski, Z. andW. F. Marzluff (2007), Gene 396(2): 373-90).

Furthermore it is preferable that the inventive nucleic acid accordingto the first aspect of the present invention does not comprise anintron.

In another particular preferred embodiment, the inventive nucleic acidsequence according to the first aspect of the present inventioncomprises or codes for from 5′ to 3′:

-   -   a) a coding region, preferably encoding at least one peptide or        protein which comprises a tumour antigen or a fragment, variant        or derivative thereof;    -   c) a poly(A) sequence; and    -   b) at least one histone stem-loop.

The inventive nucleic acid sequence according to the first embodiment ofthe present invention comprise any suitable nucleic acid, selected e.g.from any (single-stranded or double-stranded) DNA, preferably, withoutbeing limited thereto, e.g. genomic DNA, plasmid DNA, single-strandedDNA molecules, double-stranded DNA molecules, or may be selected e.g.from any PNA (peptide nucleic acid) or may be selected e.g. from any(single-stranded or double-stranded) RNA, preferably a messenger RNA(mRNA); etc. The inventive nucleic acid sequence may also comprise aviral RNA (vRNA). However, the inventive nucleic acid sequence may notbe a viral RNA or may not contain a viral RNA. More specifically, theinventive nucleic acid sequence may not contain viral sequence elements,e.g. viral enhancers or viral promotors (e.g. no inactivated viralpromoter or sequence elements, more specifically not inactivated byreplacement strategies), or other viral sequence elements, or viral orretroviral nucleic acid sequences. More specifically, the inventivenucleic acid sequence may not be a retroviral or viral vector or amodified retroviral or viral vector.

In any case, the inventive nucleic acid sequence may or may not containan enhancer and/or promoter sequence, which may be modified or not orwhich may be activated or not. The enhancer and or promoter may be plantexpressible or not expressible, and/or in eukaryotes expressible or notexpressible and/or in prokaryotes expressible or not expressible. Theinventive nucleic acid sequence may contain a sequence encoding a(self-splicing) ribozyme or not.

In specific embodiments the inventive nucleic acid sequence may be ormay comprise a self-replicating RNA (replicon).

Preferably, the inventive nucleic acid sequence is a plasmid DNA, or anRNA, particularly an mRNA.

In particular embodiments of the first aspect of the present invention,the inventive nucleic acid is a nucleic acid sequence comprised in anucleic acid suitable for in vitro transcription, particularly in anappropriate in vitro transcription vector (e.g. a plasmid or a linearnucleic acid sequence comprising specific promoters for in vitrotranscription such as T3, T7 or Sp6 promoters).

In further particular preferred embodiments of the first aspect of thepresent invention, the inventive nucleic acid is comprised in a nucleicacid suitable for transcription and/or translation in an expressionsystem (e.g. in an expression vector or plasmid), particularly aprokaryotic (e.g. bacteria like E. coli) or eukaryotic (e.g. mammaliancells like CHO cells, yeast cells or insect cells or whole organismslike plants or animals) expression system.

The term “expression system” means a system (cell culture or wholeorganisms) which is suitable for production of peptides, proteins or RNAparticularly mRNA (recombinant expression).

The inventive nucleic acid sequence according to the first aspect of thepresent invention comprises or codes for at least one histone stem-loop.In the context of the present invention, such a histone stem-loop, ingeneral (irrespective of whether it is a histone stem loop or not), istypically derived from histone genes and comprises an intramolecularbase pairing of two neighbored entirely or partially reversecomplementary sequences, thereby forming a stem-loop. A stem-loop canoccur in single-stranded DNA or, more commonly, in RNA. The structure isalso known as a hairpin or hairpin loop and usually consists of a stemand a (terminal) loop within a consecutive sequence, wherein the stem isformed by two neighbored entirely or partially reverse complementarysequences separated by a short sequence as sort of spacer, which buildsthe loop of the stem-loop structure. The two neighbored entirely orpartially reverse complementary sequences may be defined as e.g. stemloop elements stem1 and stem2. The stem loop is formed when these twoneighbored entirely or partially reverse complementary sequences, e.g.stem loop elements stem1 and stem2, form base-pairs with each other,leading to a double stranded nucleic acid sequence stretch comprising anunpaired loop at its terminal ending formed by the short sequencelocated between stem loop elements stem1 and stem2 on the consecutivesequence. The unpaired loop thereby typically represents a region of thenucleic acid which is not capable of base pairing with either of thesestem loop elements. The resulting lollipop-shaped structure is a keybuilding block of many RNA secondary structures. The formation of astem-loop structure is thus dependent on the stability of the resultingstem and loop regions, wherein the first prerequisite is typically thepresence of a sequence that can fold back on itself to form a paireddouble strand. The stability of paired stem loop elements is determinedby the length, the number of mismatches or bulges it contains (a smallnumber of mismatches is typically tolerable, especially in a long doublestranded stretch), and the base composition of the paired region. In thecontext of the present invention, a loop length of 3 to 15 bases isconceivable, while a more preferred loop length is 3-10 bases, morepreferably 3 to 8, 3 to 7, 3 to 6 or even more preferably 4 to 5 bases,and most preferably 4 bases. The stem sequence forming the doublestranded structure typically has a length of between 5 to 10 bases, morepreferably, between 5 to 8 bases.

In the context of the present invention, a histone stem-loop istypically derived from histone genes (e.g. genes from the histonefamilies H1, H2A, H2B, H3, H4) and comprises an intramolecular basepairing of two neighbored entirely or partially reverse complementarysequences, thereby forming a stem-loop. Typically, a histone 3′ UTRstem-loop is an RNA element involved in nucleocytoplasmic transport ofthe histone mRNAs, and in the regulation of stability and of translationefficiency in the cytoplasm. The mRNAs of metazoan histone genes lackpolyadenylation and a poly-A tail, instead 3′ end processing occurs at asite between this highly conserved stem-loop and a purine rich regionaround 20 nucleotides downstream (the histone downstream element, orHDE). The histone stem-loop is bound by a 31 kDa stem-loop bindingprotein (SLBP—also termed the histone hairpin binding protein, or HBP).Such histone stem-loop structures are preferably employed by the presentinvention in combination with other sequence elements and structures,which do not occur naturally (which means in untransformed livingorganisms/cells) in histone genes, but are combined—according to theinvention—to provide an artificial, heterologous nucleic acid.Accordingly, the present invention is particularly based on the findingthat an artificial (non-native) combination of a histone stem-loopstructure with other heterologous sequence elements, which do not occurin histone genes or metazoan histone genes and are isolated fromoperational and/or regulatory sequence regions (influencingtranscription and/or translation) of genes coding for proteins otherthan histones, provide advantageous effects. Accordingly, one aspect ofthe invention provides the combination of a histone stem-loop structurewith a poly(A) sequence or a sequence representing a polyadenylationsignal (3′-terminal of a coding region), which does not occur inmetazoan histone genes. According to another preferred aspect of theinvention, a combination of a histone stem-loop structure with a codingregion coding for a tumour antigenic protein, which does, preferably notoccur in metazoan histone genes, is provided herewith (coding region andhistone stem loop sequence are heterologous). It is preferred, if suchtumour antigenic proteins occur in mammalian, preferably humans, whensuffering from tumour diseases. In a still further preferred embodiment,all the elements (a), (b) and (c) of the inventive nucleic acid areheterologous to each other and are combined artificially from threedifferent sources, e.g. the (a) tumour antigenic protein coding regionfrom a human gene, (b) the histone stem loop from the untranslatedregion of a metazoan, e.g. mammalian, non-human or human, histone geneand (c) the poly(A) sequence or the polyadenylation signal from e.g. anuntranslated region of a gene other than a histone gene and other thanthe untranslated region of the tumour antigen coding region according toelement (a) of such an inventive nucleic acid.

A histone stem loop is, therefore, a stem-loop structure as describedherein, which, if preferably functionally defined, exhibits/retains theproperty of binding to its natural binding partner, the stem-loopbinding protein (SLBP—also termed the histone hairpin binding protein,or HBP).

According to the present invention the histone stem loop sequenceaccording to component (b) of claim 1 may not derived from a mousehistone protein. More specifically, the histone stem loop sequence maynot be derived from mouse histone gene H2A614. Also, the nucleic acid ofthe invention may neither contain a mouse histone stem loop sequence norcontain mouse histone gene H2A614. Further, the inventive nucleic acidsequence may not contain a stem-loop processing signal, morespecifically, a mouse histone processing signal and, most specifically,may not contain mouse stem loop processing signal H2kA614, even if theinventive nucleic acid sequence may contain at least one mammalianhistone gene. However, the at least one mammalian histone gene may notbe Seq. ID No. 7 of WO 01/12824.

According to one preferred embodiment of the first inventive aspect, theinventive nucleic acid sequence comprises or codes for at least onehistone stem-loop sequence, preferably according to at least one of thefollowing formulae (I) or (II):

formula (I) (stem-loop sequence without stem bordering elements):

formula (II) (stem-loop sequence with stem bordering elements):

wherein:

-   stem1 or stem2 bordering elements N₁₋₆ is a consecutive sequence of    1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more    preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each    N is independently from another selected from a nucleotide selected    from A, U, T, G and C, or a nucleotide analogue thereof;-   stem1 [N₀₋₂GN₃₋₅] is reverse complementary or partially reverse    complementary with element stem2, and is a consecutive sequence    between of 5 to 7 nucleotides;    -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of        0 to 1, more preferably of 1 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof;    -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of        4 to 5, more preferably of 4 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof, and    -   wherein G is guanosine or an analogue thereof, and may be        optionally replaced by a cytidine or an analogue thereof,        provided that its complementary nucleotide cytidine in stem2 is        replaced by guanosine;-   loop sequence [N₀₋₄(U/T)N₀₋₄] is located between elements stem1 and    stem2, and is a consecutive sequence of 3 to 5 nucleotides, more    preferably of 4 nucleotides;    -   wherein each N₀₋₄ is independent from another a consecutive        sequence of 0 to 4, preferably of 1 to 3, more preferably of 1        to 2 N, wherein each N is independently from another selected        from a nucleotide selected from A, U, T, G and C or a nucleotide        analogue thereof; and    -   wherein U/T represents uridine, or optionally thymidine;-   stem2 [N₃₋₅CN₀₋₂] is reverse complementary or partially reverse    complementary with element stem1, and is a consecutive sequence    between of 5 to 7 nucleotides;    -   wherein N₃₋₅ is a consecutive sequence of 3 to 5, preferably of        4 to 5, more preferably of 4 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        and C or a nucleotide analogue thereof;    -   wherein N₀₋₂ is a consecutive sequence of 0 to 2, preferably of        0 to 1, more preferably of 1 N, wherein each N is independently        from another selected from a nucleotide selected from A, U, T, G        or C or a nucleotide analogue thereof; and    -   wherein C is cytidine or an analogue thereof, and may be        optionally replaced by a guanosine or an analogue thereof        provided that its complementary nucleotide guanosine in stem1 is        replaced by cytidine;    -   wherein    -   stem1 and stem2 are capable of base pairing with each other        forming a reverse complementary sequence, wherein base pairing        may occur between stem1 and stem2, e.g. by Watson-Crick base        pairing of nucleotides A and U/T or G and C or by        non-Watson-Crick base pairing e.g. wobble base pairing, reverse        Watson-Crick base pairing, Hoogsteen base pairing, reverse        Hoogsteen base pairing or are capable of base pairing with each        other forming a partially reverse complementary sequence,        wherein an incomplete base pairing may occur between stem1 and        stem2, on the basis that one or more bases in one stem do not        have a complementary base in the reverse complementary sequence        of the other stem.

In the above context, a wobble base pairing is typically anon-Watson-Crick base pairing between two nucleotides. The four mainwobble base pairs in the present context, which may be used, areguanosine-uridine, inosine-uridine, inosine-adenosine, inosine-cytidine(G-U/T, I-U/T, I-A and I-C) and adenosine-cytidine (A-C).

Accordingly, in the context of the present invention, a wobble base is abase, which forms a wobble base pair with a further base as describedabove. Therefore non-Watson-Crick base pairing, e.g. wobble basepairing, may occur in the stem of the histone stem-loop structureaccording to the present invention.

In the above context a partially reverse complementary sequencecomprises maximally 2, preferably only one mismatch in thestem-structure of the stem-loop sequence formed by base pairing of stem1and stem2. In other words, stem1 and stem2 are preferably capable of(full) base pairing with each other throughout the entire sequence ofstem1 and stem2 (100% of possible correct Watson-Crick ornon-Watson-Crick base pairings), thereby forming a reverse complementarysequence, wherein each base has its correct Watson-Crick ornon-Watson-Crick base pendant as a complementary binding partner.Alternatively, stem1 and stem2 are preferably capable of partial basepairing with each other throughout the entire sequence of stem1 andstem2, wherein at least about 70%, 75%, 80%, 85%, 90%, or 95% of the100% possible correct Watson-Crick or non-Watson-Crick base pairings areoccupied with the correct Watson-Crick or non-Watson-Crick base pairingsand at most about 30%, 25%, 20%, 15%, 10%, or 5% of the remaining basesare unpaired.

According to a preferred embodiment of the first inventive aspect, theat least one histone stem-loop sequence (with stem bordering elements)of the inventive nucleic acid sequence as defined herein comprises alength of about 15 to about 45 nucleotides, preferably a length of about15 to about 40 nucleotides, preferably a length of about 15 to about 35nucleotides, preferably a length of about 15 to about 30 nucleotides andeven more preferably a length of about 20 to about 30 and mostpreferably a length of about 24 to about 28 nucleotides.

According to a further preferred embodiment of the first inventiveaspect, the at least one histone stem-loop sequence (without stembordering elements) of the inventive nucleic acid sequence as definedherein comprises a length of about 10 to about 30 nucleotides,preferably a length of about 10 to about 20 nucleotides, preferably alength of about 12 to about 20 nucleotides, preferably a length of about14 to about 20 nucleotides and even more preferably a length of about 16to about 17 and most preferably a length of about 16 nucleotides.

According to a further preferred embodiment of the first inventiveaspect, the inventive nucleic acid sequence according to the firstaspect of the present invention may comprise or code for at least onehistone stem-loop sequence according to at least one of the followingspecific formulae (Ia) or (IIa):

formula (IIa) (stem-loop sequence with stem bordering elements):

wherein:N, C, G, T and U are as defined above.

According to a further more particularly preferred embodiment of thefirst aspect, the inventive nucleic acid sequence may comprise or codefor at least one histone stem-loop sequence according to at least one ofthe following specific formulae (Ib) or (IIb):

formula (Ib) (stem-loop sequence without stem bordering elements):

formula (IIb) (stem-loop sequence with stem bordering elements):

wherein:

N, C, G, T and U are as defined above.

According to an even more preferred embodiment of the first inventiveaspect, the inventive nucleic acid sequence according to the firstaspect of the present invention may comprise or code for at least onehistone stem-loop sequence according to at least one of the followingspecific formulae (Ic) to (Ih) or (IIc) to (IIh), shown alternatively inits stem-loop structure and as a linear sequence representing histonestem-loop sequences as generated according to Example 1:

formula (Ic): (metazoan and protozoan histone stem-loop consensussequence without stem bordering elements):

(SEQ ID NO: 1)    N U   N   N    N-N    N-N    N-N    N-N    G-C    N-N(stem-loop structure) NGNNNNNNUNNNNNCN (linear sequence)

formula (IIc): (metazoan and protozoan histone stem-loop consensussequence with stem bordering elements):

(SEQ ID NO: 2)       N U      N   N       N-N       N-N       N-N      N-N       G-C N*N*NNNN-NNNN*N*N* (stem-loop structure)N*N*NNNNGNNNNNNUNNNNNCNNNN*N*N* (linear sequence)

formula (Id): (without stem bordering elements)

(SEQ ID NO: 3)      N U     N   N      N-N      N-N      N-N      N-N     C-G      N-N (stem-loop structure) NCNNNNNNUNNNNNGN (linearsequence)

formula (IId): (with stem bordering elements)

(SEQ ID NO: 4)       N U      N   N       N-N       N-N       N-N      N-N       C-G N*N*NNNN-NNNN*N*N* (stem-loop structure)N*N*NNNNCNNNNNNUNNNNNGNNNN*N*N* (linear sequence)

formula (Ie): (protozoan histone stem-loop consensus sequence withoutstem bordering elements)

(SEQ ID NO: 5)      N U     N   N      N-N      N-N      N-N      N-N     G-C      D-H (stem-loop structure) DGNNNNNNUNNNNNCH (linearsequence)

formula (IIe): (protozoan histone stem-loop consensus sequence with stembordering elements)

(SEQ ID NO: 6)       N U      N   N       N-N       N-N       N-N      N-N       G-C N*N*NNND-HNNN*N*N* (stem-loop structure)N*N*NNNDGNNNNNNUNNNNNCHNNN*N*N* (linear sequence)

formula (If): (metazoan histone stem-loop consensus sequence withoutstem bordering elements)

(SEQ ID NO: 7)      N U     N   N      Y-V      Y-N      B-D      N-N     G-C      N-N (stem-loop structure) NGNBYYNNUNVNDNCN (linearsequence)

formula (IIf): (metazoan histone stem-loop consensus sequence with stembordering elements)

(SEQ ID NO: 8)       N U      N   N       Y-V       Y-N       B-D      N-N       G-C N*N*NNNN-NNNN*N*N* (stem-loop structure)N*N*NNNNGNBYYNNUNVNDNCNNNN*N*N* (linear sequence)

formula (Ig): (vertebrate histone stem-loop consensus sequence withoutstem bordering elements)

(SEQ ID NO: 9)      N U     D   H      Y-A      Y-B      Y-R      H-D     G-C      N-N (stem-loop structure) NGHYYYDNUHABRDCN (linearsequence)

formula (IIg): (vertebrate histone stem-loop consensus sequence withstem bordering elements)

(SEQ ID NO: 10)       N U      D   H       Y-A       Y-B       Y-R      H-D       G-C N*N*HNNN-NNNN*N*H* (stem-loop structure)N*N*HNNNGHYYYDNUHABRDCNNNN*N*H* (linear sequence)

formula (Ih): (human histone stem-loop consensus sequence (Homo sapiens)without stem bordering elements)

(SEQ ID NO: 11)      Y U     D   H      U-A      C-S      Y-R      H-R     G-C      D-C (stem-loop structure) DGHYCUDYUHASRRCC (linearsequence)

formula (IIh): (human histone stem-loop consensus sequence (Homosapiens) with stem bordering elements)

(SEQ ID NO: 12)       Y U      D   H       U-A       C-S       Y-R      H-R       G-C N*H*AAHD-CVHB*N*H* (stem loop structure)N*H*AAHDGHYCUDYUHASRRCCVHB*N*H* (linear sequence)wherein in each of above formulae (Ic) to (Ih) or (IIc) to (IIh):

N, C, G, A, T and U are as defined above;

each U may be replaced by T;

each (highly) conserved G or C in the stem elements 1 and 2 may bereplaced by its complementary nucleotide base C or G, provided that itscomplementary nucleotide in the corresponding stem is replaced by itscomplementary nucleotide in parallel; and/or

G, A, T, U, C, R, Y, M, K, S, W, H, B, V, D, and N are nucleotide basesas defined in the following Table:

abbreviation Nucleotide bases remark G G Guanine A A Adenine T T ThymineU U Uracile C C Cytosine R G or A Purine Y T/U or C Pyrimidine M A or CAmino K G or T/U Keto S G or C Strong (3H bonds) W A or T/U Weak (2Hbonds) H A or C or T/U Not G B G or T/U or C Not A V G or C or A Not T/UD G or A or T/U Not C N G or C or T/U or A Any base * Present or notBase may be present or not

In this context it is particularly preferred that the histone stem-loopsequence according to at least one of the formulae (I) or (Ia) to (Ih)or (II) or (IIa) to (IIh) of the present invention is selected from anaturally occurring histone stem loop sequence, more particularlypreferred from protozoan or metazoan histone stem-loop sequences, andeven more particularly preferred from vertebrate and mostly preferredfrom mammalian histone stem-loop sequences especially from human histonestem-loop sequences.

According to a particularly preferred embodiment of the first aspect,the histone stem-loop sequence according to at least one of the specificformulae (I) or (Ia) to (Ih) or (II) or (IIa) to (IIh) of the presentinvention is a histone stem-loop sequence comprising at each nucleotideposition the most frequently occurring nucleotide, or either the mostfrequently or the second-most frequently occurring nucleotide ofnaturally occurring histone stem-loop sequences in metazoa and protozoa(FIG. 1), protozoa (FIG. 2), metazoa (FIG. 3), vertebrates (FIG. 4) andhumans (FIG. 5) as shown in FIG. 1-5. In this context it is particularlypreferred that at least 80%, preferably at least 85%, or most preferablyat least 90% of all nucleotides correspond to the most frequentlyoccurring nucleotide of naturally occurring histone stem-loop sequences.

In a further particular embodiment of the first aspect, the histonestem-loop sequence according to at least one of the specific formulae(I) or (Ia) to (Ih) of the present invention is selected from followinghistone stem-loop sequences (without stem-bordering elements)representing histone stem-loop sequences as generated according toExample 1:

(SEQ ID NO: 13 according to formula (Ic)) VGYYYYHHTHRVVRCB (SEQ ID NO:14 according to formula (Ic)) SGYYYTTYTMARRRCS (SEQ ID NO: 15 accordingto formula (Ic)) SGYYCTTTTMAGRRCS (SEQ ID NO: 16 according to formula(Ie)) DGNNNBNNTHVNNNCH (SEQ ID NO: 17 according to formula (Ie))RGNNNYHBTHRDNNCY (SEQ ID NO: 18 according to formula (Ie))RGNDBYHYTHRDHNCY (SEQ ID NO: 19 according to formula (If))VGYYYTYHTHRVRRCB (SEQ ID NO: 20 according to formula (If))SGYYCTTYTMAGRRCS (SEQ ID NO: 21 according to formula (If))SGYYCTTTTMAGRRCS (SEQ ID NO: 22 according to formula (Ig))GGYYCTTYTHAGRRCC (SEQ ID NO: 23 according to formula (Ig))GGCYCTTYTMAGRGCC (SEQ ID NO: 24 according to formula (Ig))GGCTCTTTTMAGRGCC (SEQ ID NO: 25 according to formula (Ih))DGHYCTDYTHASRRCC (SEQ ID NO: 26 according to formula (Ih))GGCYCTTTTHAGRGCC (SEQ ID NO: 27 according to formula (Ih))GGCYCTTTTMAGRGCC

Furthermore in this context following histone stem-loop sequences (withstem bordering elements) as generated according to Example 1 accordingto one of specific formulae (II) or (IIa) to (IIh) are particularlypreferred:

(SEQ ID NO: 28 according to formula (IIc))H*H*HHVVGYYYYHHTHRVVRCBVHH*N*N* (SEQ ID NO: 29 according to formula(IIc)) M*H*MHMSGYYYTTYTMARRRCSMCH*H*H* (SEQ ID NO: 30 according toformula (IIc)) M*M*MMMSGYYCTTTTMAGRRCSACH*M*H* (SEQ ID NO: 31 accordingto formula (IIe)) N*N*NNNDGNNNBNNTHVNNNCHNHN*N*N* (SEQ ID NO: 32according to formula (IIe)) N*N*HHNRGNNNYHBTHRDNNCYDHH*N*N* (SEQ ID NO:33 according to formula (IIe)) N*H*HHVRGNDBYHYTHRDHNCYRHH*H*H* (SEQ IDNO: 34 according to formula (IIf)) H*H*MHMVGYYYTYHTHRVRRCBVMH*H*N* (SEQID NO: 35 according to formula (IIf)) M*M*MMMSGYYCTTYTMAGRRCSMCH*H*H*(SEQ ID NO: 36 according to formula (IIf))M*M*MMMSGYYCTTTTMAGRRCSACH*M*H* (SEQ ID NO: 37 according to formula(IIg)) H*H*MAMGGYYCTTYTHAGRRCCVHN*N*M* (SEQ ID NO: 38 according toformula (IIg)) H*H*AAMGGCYCTTYTMAGRGCCVCH*H*M* (SEQ ID NO: 39 accordingto formula (IIg)) M*M*AAMGGCTCTTTTMAGRGCCMCY*M*M* (SEQ ID NO: 40according to formula (IIh)) N*H*AAHDGHYCTDYTHASRRCCVHB*N*H* (SEQ ID NO:41 according to formula (IIh)) H*H*AAMGGCYCTTTTHAGRGCCVMY*N*M* (SEQ IDNO: 42 according to formula (IIh)) H*M*AAAGGCYCTTTTMAGRGCCRMY*H*M*

According to a further preferred embodiment of the first inventiveaspect, the inventive nucleic acid sequence comprises or codes for atleast one histone stem-loop sequence showing at least about 80%,preferably at least about 85%, more preferably at least about 90%, oreven more preferably at least about 95%, sequence identity with the notto 100% conserved nucleotides in the histone stem-loop sequencesaccording to at least one of specific formulae (I) or (Ia) to (Ih) or(II) or (IIa) to (IIh) or with a naturally occurring histone stem-loopsequence.

In a preferred embodiment, the histone stem loop sequence does notcontain the loop sequence 5′-UUUC-3′. More specifically, the histonestem loop does not contain the stem1 sequence 5′-GGCUCU-3′ and/or thestem2 sequence 5′-AGAGCC-3′, respectively. In another preferredembodiment, the stem loop sequence does not contain the loop sequence5′-CCUGCCC-3′ or the loop sequence 5′-UGAAU-3′. More specifically, thestem loop does not contain the stem1 sequence 5′-CCUGAGC-3′ or does notcontain the stem1 sequence 5′-ACCUUUCUCCA-3′ and/or the stem2 sequence5′-GCUCAGG-3′ or 5′-UGGAGAAAGGU-3′, respectively. Also, as far as theinvention is not limited to histone stem loop sequences specifically,stem loop sequences are preferably not derived from a mammalian insulinreceptor 3′-untranslated region. Also, preferably, the inventive nucleicacid may not contain histone stem loop processing signals, in particularnot those derived from mouse histone gene H2A614 gene (H2kA614).

The inventive nucleic acid sequence according to the first aspect of thepresent invention may optionally comprise or code for a poly(A)sequence. When present, such a poly(A) sequence comprises a sequence ofabout 25 to about 400 adenosine nucleotides, preferably a sequence ofabout 30 or, more preferably, of about 50 to about 400 adenosinenucleotides, more preferably a sequence of about 50 to about 300adenosine nucleotides, even more preferably a sequence of about 50 toabout 250 adenosine nucleotides, most preferably a sequence of about 60to about 250 adenosine nucleotides. In this context the term “about”refers to a deviation of ±10% of the value(s) it is attached to.Accordingly, the poly(A) sequence contains at least 25 or more than 25,more preferably, at least 30, more preferably at least 50 adenosinenucleotides. Therefore, such a poly (A) sequence does typically notcontain less than 20 adenosine nucleotides. More particularly, it doesnot contain 10 and/or less than 10 adenosine nucleotides.

Preferably, the nucleic acid according of the present invention does notcontain one or two or at least one or all but one or all of thecomponents of the group consisting of: a sequence encoding a ribozyme(preferably a self-splicing ribozyme), a viral nucleic acid sequence, ahistone stem-loop processing signal, in particular a histone-stem loopprocessing sequence derived from mouse histone H2A614 gene, a Neo gene,an inactivated promoter sequence and an inactivated enhancer sequence.Even more preferably, the nucleic acid according to the invention doesnot contain a ribozyme, preferably a self-splicing ribozyme, and one ofthe group consisting of: a Neo gene, an inactivated promoter sequence,an inactivated enhancer sequence, a histone stem-loop processing signal,in particular a histone-stem loop processing sequence derived from mousehistone H2A614 gene. Accordingly, the nucleic acid may in a preferredmode neither contain a ribozyme, preferably a self-splicing ribozyme,nor a Neo gene or, alternatively, neither a ribozyme, preferably aself-splicing ribozyme, nor any resistance gene (e.g. usually appliedfor selection). In another preferred mode, the nucleic acid of theinvention may neither contain a ribozyme, preferably a self-splicingribozyme nor a histone stem-loop processing signal, in particular ahistone-stem loop processing sequence derived from mouse histone H2A614gene

Alternatively, according to the first aspect of the present invention,the inventive nucleic sequence optionally comprises a polyadenylationsignal which is defined herein as a signal which conveys polyadenylationto a (transcribed) mRNA by specific protein factors (e.g. cleavage andpolyadenylation specificity factor (CPSF), cleavage stimulation factor(CstF), cleavage factors I and II (CF I and CF II), poly(A) polymerase(PAP)). In this context a consensus polyadenylation signal is preferredcomprising the NN(U/T)ANA consensus sequence. In a particular preferredaspect the polyadenylation signal comprises one of the followingsequences: AA(U/T)AAA or A(U/T)(U/T)AAA (wherein uridine is usuallypresent in RNA and thymidine is usually present in DNA). In someembodiments, the polyadenylation signal used in the inventive nucleicacid does not correspond to the U3 snRNA, U5, the polyadenylationprocessing signal from human gene G-CSF, or the SV40 polyadenylationsignal sequences. In particular, the above polyadenylation signals arenot combined with any antibiotics resistance gene (or any otherreporter, marker or selection gene), in particular not with theresistance neo gene (neomycin phosphotransferase) (as the gene of thecoding region according to element (a) of the inventive nucleic acid insuch an inventive nucleic acid. And any of the above polyadenylationsignals are preferably not combined with the histone stem loop or thehistone stem loop processing signal from mouse histone gene H2A614 in aninventive nucleic acid.

The inventive nucleic acid sequence according to the first aspect of thepresent invention may furthermore encode a protein or a peptide, whichcomprises a tumour antigen or a fragment, variant or derivative thereof.

Tumour antigens are preferably located on the surface of the (tumour)cell characterizing a mammalian, in particular human tumour (in e.g.systemic or solid tumour diseases). Tumour antigens may also be selectedfrom proteins, which are overexpressed in tumour cells compared to anormal cell. Furthermore, tumour antigens also includes antigensexpressed in cells which are (were) not themselves (or originally notthemselves) degenerated but are associated with the supposed tumour.Antigens which are connected with tumour-supplying vessels or(re)formation thereof, in particular those antigens which are associatedwith neovascularization, e.g. growth factors, such as VEGF, bFGF etc.,are also included herein. Antigens connected with a tumour furthermoreinclude antigens from cells or tissues, typically embedding the tumour.Further, some substances (usually proteins or peptides) are expressed inpatients suffering (knowingly or not-knowingly) from a cancer diseaseand they occur in increased concentrations in the body fluids of saidpatients. These substances are also referred to as “tumour antigens”,however they are not antigens in the stringent meaning of an immuneresponse inducing substance. The class of tumour antigens can be dividedfurther into tumour-specific antigens (TSAs) andtumour-associated-antigens (TAAs). TSAs can only be presented by tumourcells and never by normal “healthy” cells. They typically result from atumour specific mutation. TAAs, which are more common, are usuallypresented by both tumour and healthy cells. These antigens arerecognized and the antigen-presenting cell can be destroyed by cytotoxicT cells. Additionally, tumour antigens can also occur on the surface ofthe tumour in the form of, e.g., a mutated receptor. In this case, theycan be recognized by antibodies.

Further, tumour associated antigens may be classified as tissue-specificantigens, also called melanocyte-specific antigens, cancer-testisantigens and tumour-specific antigens. Cancer-testis antigens aretypically understood to be peptides or proteins of germ-line associatedgenes which may be activated in a wide variety of tumours. Humancancer-testis antigens may be further subdivided into antigens which areencoded on the X chromosome, so-called CT-X antigens, and those antigenswhich are not encoded on the X chromosome, the so-called (non-X CTantigens). Cancer-testis antigens which are encoded on the X-chromosomecomprises, for example, the family of melanoma antigen genes, theso-called MAGE-family. The genes of the MAGE-family may be characterisedby a shared MAGE homology domain (MHD). Each of these antigens, i.e.melanocyte-specific antigens, cancer-testis antigens and tumour-specificantigens, may elicit autologous cellular and humoral immune response.Accordingly, the tumour antigen encoded by the inventive nucleic acidsequence is preferably a melanocyte-specific antigen, a cancer-testisantigen or a tumour-specific antigens, preferably it may be a CT-Xantigen, a non-X CT-antigens, a binding partner for a CT-X antigen or abinding partner for a non-X CT-antigen or a tumour-specific antigen,more preferably a CT-X antigen, a binding partner for a non-X CT-antigenor a tumour-specific antigen.

Particular preferred tumour antigens are selected from the listconsisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1,alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m,alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, BAGE-1,BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsinB, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55,CD56, CDso, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66,COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten,cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m,EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphAs, ErbBs, ETV6-AML1, EZH2,FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE7b, GAGE-8, GDEP, GnT-V, gploo, GPC3, GPNMB/m, HAGE, HAST-2, hepsin,Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE,homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-s5, HPV-E6, HPV-E7, HSP70-2M,HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature lamininreceptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3,MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1,MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1,MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrixprotein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin classI/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m,NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-B, NY-ESO-1, OA1,OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE,PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1,survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP,TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1,TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, andWT1. Such tumour antigens preferably may be selected from the groupconsisting of p53, CA125, EGFR, Her2/neu, hTERT, PAP, MAGE-A1, MAGE-A3,Mesothelin, MUC-1, GP100, MART-1, Tyrosinase, PSA, PSCA, PSMA, STEAP-1,VEGF, VEGFR1, VEGFR2, Ras, CEA or WT1, and more preferably from PAP,MAGE-A3, WT1, and MUC-1. Such tumour antigens preferably may be selectedfrom the group consisting of MAGE-A1 (e.g. MAGE-A1 according toaccession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g. MAGE-A6according to accession number NM_005363), MAGE-C1, MAGE-C2, melan-A(e.g. melan-A according to accession number NM_005511), GP100 (e.g.GP100 according to accession number M77348), tyrosinase (e.g. tyrosinaseaccording to accession number NM_000372), surviving (e.g. survivinaccording to accession number AF077350), CEA (e.g. CEA according toaccession number NM_004363), Her-2/neu (e.g. Her-2/neu according toaccession number M11730), WT1 (e.g. WT1 according to accession numberNM_000378), PRAME (e.g. PRAME according to accession number NM_006115),EGFRI (epidermal growth factor receptor 1) (e.g. EGFRI (epidermal growthfactor receptor 1) according to accession number AF288738), MUC1,mucin-1 (e.g. mucin-1 according to accession number NM_002456), SEC61G(e.g. SEC61G according to accession number NM_014302), hTERT (e.g. hTERTaccession number NM_198253), 5T4 (e.g. 5T4 according to accession numberNM_006670), TRP-2 (e.g. TRP-2 according to accession number NM_001922),STEAP1, PCA, PSA, PSMA, etc.

Furthermore tumour antigens also may encompass idiotypic antigensassociated with a cancer or tumour disease, particularly lymphoma or alymphoma associated disease, wherein said idiotypic antigen is animmunoglobulin idiotype of a lymphoid blood cell or a T cell receptoridiotype of a lymphoid blood cell.

Tumour antigenic proteins for the treatment of cancer or tumourdiseases, are typically proteins of mammalian origin, preferably ofhuman origin. Their selection for treatment of the subject depends onthe tumour type to be treated and the expression profile of theindividual tumour. A human suffering from prostate cancer, is e.g.preferably treated by a tumour antigen, which is typically expressed (oroverexpressed) in prostate carcinoma or specifically overexpressed inthe subject to be treated, e.g. any of PSMA, PSCA, and/or PSA.

The coding region of the inventive nucleic acid according to the firstaspect of the present invention may occur as a mono-, di-, or evenmulticistronic nucleic acid, i.e. a nucleic acid which carries thecoding sequences of one, two or more proteins or peptides. Such codingsequences in di-, or even multicistronic nucleic acids may be separatedby at least one internal ribosome entry site (IRES) sequence, e.g. asdescribed herein or by signal peptides which induce the cleavage of theresulting polypeptide which comprises several proteins or peptides.

According to the first aspect of the present invention, the inventivenucleic acid sequence comprises a coding region, encoding a peptide orprotein which comprises a tumour antigen or a fragment, variant orderivative thereof. Preferably, the encoded tumour antigen is no histoneprotein. In the context of the present invention such a histone proteinis typically a strongly alkaline protein found in eukaryotic cellnuclei, which package and order the DNA into structural units callednucleosomes. Histone proteins are the chief protein components ofchromatin, act as spools around which DNA winds, and play a role in generegulation. Without histones, the unwound DNA in chromosomes would bevery long (a length to width ratio of more than 10 million to one inhuman DNA). For example, each human cell has about 1.8 meters of DNA,but wound on the histones it has about 90 millimeters of chromatin,which, when duplicated and condensed during mitosis, result in about 120micrometers of chromosomes. More preferably, in the context of thepresent invention such a histone protein is typically defined as ahighly conserved protein selected from one of the following five majorclasses of histones: H1/H5, H2A, H2B, H3, and H4″, preferably selectedfrom mammalian histone, more preferably from human histones or histoneproteins. Such histones or histone proteins are typically organised intotwo super-classes defined as core histones, comprising histones H2A,H2B, H3 and H4, and linker histones, comprising histones H1 and H5.

In this context, linker histones, preferably excluded from the scope ofprotection of the pending invention, preferably mammalian linkerhistones, more preferably human linker histones, are typically selectedfrom H1, including H1F, particularly including H1F0, H1FNT, H1FOO, H1FX,and H1H1, particularly including HIST1H1A, HIST1H1B, HIST1H1C, HIST1H1D,HIST1H1E, HIST1H1T; and Furthermore, core histones, preferably excludedfrom the scope of protection of the pending invention, preferablymammalian core histones, more preferably human core histones, aretypically selected from H2A, including H2AF, particularly includingH2AFB1, H2AFB2, H2AFB3, H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2, H2AFZ, andH2A1, particularly including HIST1H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD,HIST1H2AE, HIST1H2AG, HIST1H2AI, HIST1H2AJ, HIST1H2AK, HIST1H2AL,HIST1H2AM, and H2A2, particularly including HIST2H2AA3, HIST2H2AC; H2B,including H2BF, particularly including H2BFM, H2BFO, H2BFS, H2BFWT H2B1,particularly including HIST1H2BA, HIST1H2BB, HIST1H2BC, HIST1H2BD,HIST1H2BE, HIST1H2BF, HIST1H2BG, HIST1H2BH, HIST1H2BI, HIST1H2BJ,HIST1H2BK, HIST1H2BL, HIST1H2BM, HIST1H2BN, HIST1H2BO, and H2B2,particularly including HIST2H2BE; H3, including H3A1, particularlyincluding HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F,HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J, and H3A2, particularly includingHIST2H3C, and H3A3, particularly including HIST3H3; H4, including H41,particularly including HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E,HIST1H4F, HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L,and H44, particularly including HIST4H4, and H5.

According to the first aspect of the present invention, the inventivenucleic acid sequence comprises a coding region, encoding a peptide orprotein which comprises a tumour antigen or a fragment, variant orderivative thereof. Preferably, the encoded tumour antigen is noreporter protein (e.g. Luciferase, Green Fluorescent Protein (GFP),Enhanced Green Fluorescent Protein (EGFP), β-Galactosidase) and nomarker or selection protein (e.g. alpha-Globin, Galactokinase andXanthine:guanine phosphoribosyl transferase (GPT)). Preferably, thenucleic acid sequence of the invention does not contain a (bacterial)antibiotics resistance gene, in particular not a neo gene sequence(Neomycin resistance gene) or CAT gene sequence (chloramphenicol acetyltransferase, chloramphenicol resistance gene).

The inventive nucleic acid as define above, comprises or codes for a) acoding region, encoding a peptide or protein which comprises a tumourantigen or a fragment, variant or derivative thereof; b) at least onehistone stem-loop, and c) a poly(A) sequence or polyadenylation signal;preferably for increasing the expression of said encoded peptide orprotein, wherein the encoded peptide or protein is preferably no histoneprotein, no reporter protein and/or no marker or selection protein, asdefined above. The elements b) to c) of the inventive nucleic acid mayoccur in the inventive nucleic acid in any order, i.e. the elements a),b) and c) may occur in the order a), b) and c) or a), c) and b) from 5′to 3′ direction in the inventive nucleic acid sequence, wherein furtherelements as described herein, may also be contained, such as a 5′-CAPstructure, a poly(C) sequence, stabilization sequences, IRES sequences,etc. Each of the elements a) to c) of the inventive nucleic acid,particularly a) in di- or multicistronic constructs and/or each of theelements b) and c), more preferably element b) may also be repeated atleast once, preferably twice or more in the inventive nucleic acid. Asan example, the inventive nucleic acid may show its sequence elementsa), b) and optionally c) in e.g. the following order:

5′-coding region-histone stem-loop-poly(A) sequence-3′; or 5′-codingregion-histone stem-loop-polyadenylation signal-3′; or 5′-codingregion-poly(A) sequence-histone stem-loop-3′; or 5′-codingregion-polyadenylation signal-histone stem-loop-3′; or 5′-codingregion-coding region-histone stem-loop-polyadenylation signal-3′; or5′-coding region-histone stem-loop-histone stem-loop-poly(A)sequence-3′; or 5′-coding region-histone stem-loop-histonestem-loop-polyadenylation signal-3′; etc.

In this context it is particularly preferred that the inventive nucleicacid sequence comprises or codes for a) a coding region, encoding apeptide or protein which comprises a tumour antigen or fragment, variantor derivative thereof; b) at least one histone stem-loop, and c) apoly(A) sequence or polyadenylation sequence; preferably for increasingthe expression level of said encoded peptide or protein, wherein theencoded protein is preferably no histone protein, no reporter protein(e.g. Luciferase, GFP, EGFP, β-Galactosidase, particularly EGFP) and/orno marker or selection protein (e.g. alpha-Globin, Galactokinase andXanthine:Guanine phosphoribosyl transferase (GPT)).

In a further preferred embodiment of the first aspect the inventivenucleic acid sequence as defined herein may also occur in the form of amodified nucleic acid.

In this context, the inventive nucleic acid sequence as defined hereinmay be modified to provide a “stabilized nucleic acid”, preferably astabilized RNA, more preferably an RNA that is essentially resistant toin vivo degradation (e.g. by an exo- or endo-nuclease). A stabilizednucleic acid may e.g. be obtained by modification of the G/C content ofthe coding region of the inventive nucleic acid sequence, byintroduction of nucleotide analogues (e.g. nucleotides with backbonemodifications, sugar modifications or base modifications) or byintroduction of stabilization sequences in the 3′- and/or5′-untranslated region of the inventive nucleic acid sequence.

As mentioned above, the inventive nucleic acid sequence as definedherein may contain nucleotide analogues/modifications e.g. backbonemodifications, sugar modifications or base modifications. A backbonemodification in connection with the present invention is a modificationin which phosphates of the backbone of the nucleotides contained ininventive nucleic acid sequence as defined herein are chemicallymodified. A sugar modification in connection with the present inventionis a chemical modification of the sugar of the nucleotides of theinventive nucleic acid sequence as defined herein. Furthermore, a basemodification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides of the nucleic acidmolecule of the inventive nucleic acid sequence. In this contextnucleotide analogues or modifications are preferably selected fromnucleotide analogues which are applicable for transcription and/ortranslation.

In a particular preferred embodiment of the first aspect of the presentinvention the herein defined nucleotide analogues/modifications areselected from base modifications which additionally increase theexpression of the encoded protein and which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-iodouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate,5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate,6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

According to a further embodiment, the inventive nucleic acid sequenceas defined herein can contain a lipid modification. Such alipid-modified nucleic acid typically comprises a nucleic acid asdefined herein. Such a lipid-modified nucleic acid molecule of theinventive nucleic acid sequence as defined herein typically furthercomprises at least one linker covalently linked with that nucleic acidmolecule, and at least one lipid covalently linked with the respectivelinker. Alternatively, the lipid-modified nucleic acid moleculecomprises at least one nucleic acid molecule as defined herein and atleast one (bifunctional) lipid covalently linked (without a linker) withthat nucleic acid molecule. According to a third alternative, thelipid-modified nucleic acid molecule comprises a nucleic acid moleculeas defined herein, at least one linker covalently linked with thatnucleic acid molecule, and at least one lipid covalently linked with therespective linker, and also at least one (bifunctional) lipid covalentlylinked (without a linker) with that nucleic acid molecule. In thiscontext it is particularly preferred that the lipid modification ispresent at the terminal ends of a linear inventive nucleic acidsequence.

According to another preferred embodiment of the first aspect of theinvention, the inventive nucleic acid sequence as defined herein,particularly if provided as an (m)RNA, can therefore be stabilizedagainst degradation by RNases by the addition of a so-called “5′ CAP”structure.

According to a further preferred embodiment of the first aspect of theinvention, the inventive nucleic acid sequence as defined herein can bemodified by a sequence of at least 10 cytidines, preferably at least 20cytidines, more preferably at least 30 cytidines (so-called “poly(C)sequence”). Particularly, the inventive nucleic acid sequence maycontain or code for a poly(C) sequence of typically about 10 to 200cytidine nucleotides, preferably about 10 to 100 cytidine nucleotides,more preferably about 10 to 70 cytidine nucleotides or even morepreferably about 20 to 50 or even 20 to 30 cytidine nucleotides. Thispoly(C) sequence is preferably located 3′ of the coding region comprisedin the inventive nucleic acid according to the first aspect of thepresent invention.

In a particularly preferred embodiment of the present invention, the G/Ccontent of the coding region, encoding at least one peptide or proteinwhich comprises a tumour antigen or a fragment, variant or derivativethereof of the inventive nucleic acid sequence as defined herein, ismodified, particularly increased, compared to the G/C content of itsparticular wild type coding region, i.e. the unmodified coding region.The encoded amino acid sequence of the coding region is preferably notmodified compared to the coded amino acid sequence of the particularwild type coding region.

The modification of the G/C-content of the coding region of theinventive nucleic acid sequence as defined herein is based on the factthat the sequence of any mRNA region to be translated is important forefficient translation of that mRNA. Thus, the composition and thesequence of various nucleotides are important. In particular, mRNAsequences having an increased G (guanosine)/C (cytosine) content aremore stable than mRNA sequences having an increased A (adenosine)/U(uracil) content. According to the invention, the codons of the codingregion are therefore varied compared to its wild type coding region,while retaining the translated amino acid sequence, such that theyinclude an increased amount of G/C nucleotides. In respect to the factthat several codons code for one and the same amino acid (so-calleddegeneration of the genetic code), the most favourable codons for thestability can be determined (so-called alternative codon usage).

Depending on the amino acid to be encoded by the coding region of theinventive nucleic acid sequence as defined herein, there are variouspossibilities for modification of the nucleic acid sequence, e.g. thecoding region, compared to its wild type coding region. In the case ofamino acids which are encoded by codons which contain exclusively G or Cnucleotides, no modification of the codon is necessary. Thus, the codonsfor Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC orGGG) require no modification, since no A or U is present.

In contrast, codons which contain A and/or U nucleotides can be modifiedby substitution of other codons which code for the same amino acids butcontain no A and/or U. Examples of these are:

the codons for Pro can be modified from CCU or CCA to CCC or CCG;

the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGCor CGG;

the codons for Ala can be modified from GCU or GCA to GCC or GCG;

the codons for Gly can be modified from GGU or GGA to GGC or GGG.

In other cases, although A or U nucleotides cannot be eliminated fromthe codons, it is however possible to decrease the A and U content byusing codons which contain a lower content of A and/or U nucleotides.Examples of these are:

the codons for Phe can be modified from UUU to UUC;

the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC orCUG;

the codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG orAGC;

the codon for Tyr can be modified from UAU to UAC;

the codon for Cys can be modified from UGU to UGC;

the codon for His can be modified from CAU to CAC;

the codon for Gln can be modified from CAA to CAG;

the codons for Ile can be modified from AUU or AUA to AUC;

the codons for Thr can be modified from ACU or ACA to ACC or ACG;

the codon for Asn can be modified from AAU to AAC;

the codon for Lys can be modified from AAA to AAG;

the codons for Val can be modified from GUU or GUA to GUC or GUG;

the codon for Asp can be modified from GAU to GAC;

the codon for Glu can be modified from GAA to GAG;

the stop codon UAA can be modified to UAG or UGA.

In the case of the codons for Met (AUG) and Trp (UGG), on the otherhand, there is no possibility of sequence modification.

The substitutions listed above can be used either individually or in allpossible combinations to increase the G/C content of the coding regionof the inventive nucleic acid sequence as defined herein, compared toits particular wild type coding region (i.e. the original sequence).Thus, for example, all codons for Thr occurring in the wild typesequence can be modified to ACC (or ACG).

In the above context, mRNA codons are shown. Therefore uridine presentin an mRNA may also be present as thymidine in the respective DNA codingfor the particular mRNA.

Preferably, the G/C content of the coding region of the inventivenucleic acid sequence as defined herein is increased by at least 7%,more preferably by at least 15%, particularly preferably by at least20%, compared to the G/C content of the wild type coding region.According to a specific embodiment at least 5%, 10%, 20%, 30%, 40%, 50%,60%, more preferably at least 70%, even more preferably at least 80% andmost preferably at least 90%, 95% or even 100% of the substitutablecodons in the coding region encoding at least one peptide or proteinwhich comprises a tumour antigen or a fragment, variant or derivativethereof are substituted, thereby increasing the G/C content of saidcoding region.

In this context, it is particularly preferable to increase the G/Ccontent of the coding region of the inventive nucleic acid sequence asdefined herein, to the maximum (i.e. 100% of the substitutable codons),compared to the wild type coding region.

According to the invention, a further preferred modification of thecoding region encoding at least one peptide or protein which comprises atumour antigen or a fragment, variant or derivative thereof of theinventive nucleic acid sequence as defined herein, is based on thefinding that the translation efficiency is also determined by adifferent frequency in the occurrence of tRNAs in cells. Thus, ifso-called “rare codons” are present in the coding region of theinventive nucleic acid sequence as defined herein, to an increasedextent, the corresponding modified nucleic acid sequence is translatedto a significantly poorer degree than in the case where codons codingfor relatively “frequent” tRNAs are present.

In this context the coding region of the inventive nucleic acid sequenceis preferably modified compared to the corresponding wild type codingregion such that at least one codon of the wild type sequence whichcodes for a tRNA which is relatively rare in the cell is exchanged for acodon which codes for a tRNA which is relatively frequent in the celland carries the same amino acid as the relatively rare tRNA. By thismodification, the coding region of the inventive nucleic acid sequenceas defined herein, is modified such that codons for which frequentlyoccurring tRNAs are available are inserted. In other words, according tothe invention, by this modification all codons of the wild type codingregion which code for a tRNA which is relatively rare in the cell can ineach case be exchanged for a codon which codes for a tRNA which isrelatively frequent in the cell and which, in each case, carries thesame amino acid as the relatively rare tRNA.

Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Thecodons which use for the particular amino acid the tRNA which occurs themost frequently, e.g. the Gly codon, which uses the tRNA which occursthe most frequently in the (human) cell, are particularly preferred.

According to the invention, it is particularly preferable to link thesequential G/C content which is increased, in particular maximized, inthe coding region of the inventive nucleic acid sequence as definedherein, with the “frequent” codons without modifying the amino acidsequence of the peptide or protein encoded by the coding region of thenucleic acid sequence. This preferred embodiment allows provision of aparticularly efficiently translated and stabilized (modified) inventivenucleic acid sequence as defined herein.

According to another preferred embodiment of the first aspect of theinvention, the inventive nucleic acid sequence as defined herein,preferably has additionally at least one 5′ and/or 3′ stabilizingsequence. These stabilizing sequences in the 5′ and/or 3′ untranslatedregions have the effect of increasing the half-life of the nucleic acid,particularly of the mRNA in the cytosol. These stabilizing sequences canhave 100% sequence identity to naturally occurring sequences which occurin viruses, bacteria and eukaryotes, but can also be partly orcompletely synthetic. The untranslated sequences (UTR) of the(alpha-)globin gene, e.g. from Homo sapiens or Xenopus laevis may bementioned as an example of stabilizing sequences which can be used inthe present invention for a stabilized nucleic acid. Another example ofa stabilizing sequence has the general formula(C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC (SEQ ID NO: 55), which is containedin the 3′-UTRs of the very stable RNAs which code for (alpha-)globin,type(I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf.Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Suchstabilizing sequences can of course be used individually or incombination with one another and also in combination with otherstabilizing sequences known to a person skilled in the art. In thiscontext it is particularly preferred that the 3′ UTR sequence of thealpha globin gene is located 3′ of the coding region encoding at leastone peptide or protein which comprises a tumour antigen or a fragment,variant or derivative thereof comprised in the inventive nucleic acidsequence according to the first aspect of the present invention.

Substitutions, additions or eliminations of bases are preferably carriedout with the inventive nucleic acid sequence as defined herein, using aDNA matrix for preparation of the nucleic acid sequence by techniques ofthe well-known site directed mutagenesis or with an oligonucleotideligation strategy (see e.g. Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., ColdSpring Harbor, N.Y., 2001).

Any of the above modifications may be applied to the inventive nucleicacid sequence as defined herein and further to any nucleic acid as usedin the context of the present invention and may be, if suitable ornecessary, be combined with each other in any combination, provided,these combinations of modifications do not interfere with each other inthe respective nucleic acid. A person skilled in the art will be able totake his choice accordingly.

Nucleic acid sequences used according to the present invention asdefined herein may be prepared using any method known in the art,including synthetic methods such as e.g. solid phase synthesis, as wellas in vitro methods, such as in vitro transcription reactions or in vivoreactions, such as in vivo propagation of DNA plasmids in bacteria.

In such a process, for preparation of the inventive nucleic acidsequence as defined herein, especially if the nucleic acid is in theform of an mRNA, a corresponding DNA molecule may be transcribed invitro. This DNA matrix preferably comprises a suitable promoter, e.g. aT7 or SP6 promoter, for in vitro transcription, which is followed by thedesired nucleotide sequence for the nucleic acid molecule, e.g. mRNA, tobe prepared and a termination signal for in vitro transcription. The DNAmolecule, which forms the matrix of the at least one RNA of interest,may be prepared by fermentative proliferation and subsequent isolationas part of a plasmid which can be replicated in bacteria. Plasmids whichmay be mentioned as suitable for the present invention are e.g. theplasmids pT7Ts (GENBANK® genetic sequence database accession numberU26404; Lai et al., Development 1995, 121: 2349 to 2360), pGEM® series,e.g. pGEM®-1 (GENBANK® genetic sequence database accession numberX65300; from Promega) and pSP64 (GENBANK® genetic sequence databaseaccession number X65327); cf. also Mezei and Storts, Purification of PCRProducts, in: Griffin and Griffin (ed.), PCR Technology: CurrentInnovation, CRC Press, Boca Raton, Fla., 2001.

The inventive nucleic acid sequence as defined herein as well asproteins or peptides as encoded by this nucleic acid sequence maycomprise fragments or variants of those sequences. Such fragments orvariants may typically comprise a sequence having a sequence identitywith one of the above mentioned nucleic acids, or with one of theproteins or peptides or sequences, if encoded by the inventive nucleicacid sequence, of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferablyat least 70%, more preferably at least 80%, equally more preferably atleast 85%, even more preferably at least 90% and most preferably atleast 95% or even 97%, 98% or 99%, to the entire wild type sequence,either on nucleic acid level or on amino acid level.

“Fragments” of proteins or peptides in the context of the presentinvention (e.g. as encoded by the inventive nucleic acid sequence asdefined herein) may comprise a sequence of a protein or peptide asdefined herein, which is, with regard to its amino acid sequence (or itsencoded nucleic acid molecule), N-terminally, C-terminally and/orintrasequentially truncated/shortened compared to the amino acidsequence of the original (native) protein (or its encoded nucleic acidmolecule). Such truncation may thus occur either on the amino acid levelor correspondingly on the nucleic acid level. A sequence identity withrespect to such a fragment as defined herein may therefore preferablyrefer to the entire protein or peptide as defined herein or to theentire (coding) nucleic acid molecule of such a protein or peptide.Likewise, “fragments” of nucleic acids in the context of the presentinvention may comprise a sequence of a nucleic acid as defined herein,which is, with regard to its nucleic acid molecule 5′-, 3′- and/orintrasequentially truncated/shortened compared to the nucleic acidmolecule of the original (native) nucleic acid molecule. A sequenceidentity with respect to such a fragment as defined herein may thereforepreferably refer to the entire nucleic acid as defined herein and thepreferred sequence identity level typically is as indicated herein.Fragments have the same biological function or specific activity or atleast retain an activity of the natural full length protein of at least50%, more preferably at least 70%, even more preferably at least 90%(measured in an appropriate functional assay, e.g. an assay assessingthe antigenic property by a appropriate assay system which e.g. measuresan immunological reaction, e.g. expression and/or secretion of anappropriate cytokine (as an indicator of the immune reaction)) ascompared to the full-length native peptide or protein, e.g. its specificantigenic or therapeutic property. Accordingly, in a preferredembodiment, the “fragment” is a portion of the full-length (naturallyoccurring) tumour antigenic protein, which exerts tumour antigenicproperties on the immune system as indicated herein.

Fragments of proteins or peptides in the context of the presentinvention (e.g. as encoded by the inventive nucleic acid sequence asdefined herein) may furthermore comprise a sequence of a protein orpeptide as defined herein, which has a length of about 6 to about 20 oreven more amino acids, e.g. fragments as processed and presented by MHCclass I molecules, preferably having a length of about 8 to about 10amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12 amino acids), orfragments as processed and presented by MHC class II molecules,preferably having a length of about 13 or more amino acids, e.g. 13, 14,15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragmentsmay be selected from any part of the amino acid sequence. Thesefragments are typically recognized by T-cells in form of a complexconsisting of the peptide fragment and an MHC molecule, i.e. thefragments are typically not recognized in their native form. Fragmentsof proteins or peptides as defined herein may comprise at least oneepitope of those proteins or peptides. Furthermore, also domains of aprotein, like the extracellular domain, the intracellular domain or thetransmembrane domain and shortened or truncated versions of a proteinmay be understood to comprise a fragment of a protein.

Fragments of proteins or peptides as defined herein (e.g. as encoded bythe inventive nucleic acid sequence as defined herein) may also compriseepitopes of those proteins or peptides. T cell epitopes or parts of theproteins in the context of the present invention may comprise fragmentspreferably having a length of about 6 to about 20 or even more aminoacids, e.g. fragments as processed and presented by MHC class Imolecules, preferably having a length of about 8 to about 10 aminoacids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragmentsas processed and presented by MHC class II molecules, preferably havinga length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18,19, 20 or even more amino acids, wherein these fragments may be selectedfrom any part of the amino acid sequence. These fragments are typicallyrecognized by T cells in form of a complex consisting of the peptidefragment and an MHC molecule, i.e. the fragments are typically notrecognized in their native form.

B cell epitopes are typically fragments located on the outer surface of(native) protein or peptide antigens as defined herein, preferablyhaving 5 to 15 amino acids, more preferably having 5 to 12 amino acids,even more preferably having 6 to 9 amino acids, which may be recognizedby antibodies, i.e. in their native form.

Such epitopes of proteins or peptides may furthermore be selected fromany of the herein mentioned variants of such proteins or peptides. Inthis context antigenic determinants can be conformational ordiscontinuous epitopes which are composed of segments of the proteins orpeptides as defined herein that are discontinuous in the amino acidsequence of the proteins or peptides as defined herein but are broughttogether in the three-dimensional structure or continuous or linearepitopes which are composed of a single polypeptide chain.

“Variants” of proteins or peptides as defined in the context of thepresent invention may be encoded by the inventive nucleic acid sequenceas defined herein. Thereby, a protein or peptide may be generated,having an amino acid sequence which differs from the original sequencein one or more (2, 3, 4, 5, 6, 7 or more) mutation(s), such as one ormore substituted, inserted and/or deleted amino acid(s). The preferredlevel of sequence identity of “variants” in view of the full-lengthnatural protein sequence typically is as indicated herein. Preferably,variants have the same biological function or specific activity or atleast retain an activity of the natural full length protein of at least50%, more preferably at least 70%, even more preferably at least 90%(measured in an appropriate functional assay, e.g. by an assay assessingthe immunological reaction towards the tumour antigen by the secretionand/or expression of one or more cytokines) compared to the full-lengthnative peptide or protein, e.g. its specific antigenic property.Accordingly, in a preferred embodiment, the “variant” is a variant of atumour antigenic protein, which exerts tumour antigenic properties tothe extent as indicated herein.

“Variants” of proteins or peptides as defined in the context of thepresent invention (e.g. as encoded by a nucleic acid as defined herein)may comprise conservative amino acid substitution(s) compared to theirnative, i.e. non-mutated physiological, sequence. Those amino acidsequences as well as their encoding nucleotide sequences in particularfall under the term variants as defined herein. Substitutions in whichamino acids, which originate from the same class, are exchanged for oneanother are called conservative substitutions. In particular, these areamino acids having aliphatic side chains, positively or negativelycharged side chains, aromatic groups in the side chains or amino acids,the side chains of which can enter into hydrogen bridges, e.g. sidechains which have a hydroxyl function. This means that e.g. an aminoacid having a polar side chain is replaced by another amino acid havinga likewise polar side chain, or, for example, an amino acidcharacterized by a hydrophobic side chain is substituted by anotheramino acid having a likewise hydrophobic side chain (e.g. serine(threonine) by threonine (serine) or leucine (isoleucine) by isoleucine(leucine)). Insertions and substitutions are possible, in particular, atthose sequence positions which cause no modification to thethree-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) ordeletion(s) can easily be determined e.g. using CD spectra (circulardichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORDof Polypeptides, in: Modern Physical Methods in Biochemistry, Neubergeret al. (ed.), Elsevier, Amsterdam).

Furthermore, variants of proteins or peptides as defined herein, whichmay be encoded by the inventive nucleic acid sequence as defined herein,may also comprise those sequences, wherein nucleotides of the nucleicacid are exchanged according to the degeneration of the genetic code,without leading to an alteration of the respective amino acid sequenceof the protein or peptide, i.e. the amino acid sequence or at least partthereof may not differ from the original sequence in one or moremutation(s) within the above meaning.

In order to determine the percentage to which two sequences areidentical, e.g. nucleic acid sequences or amino acid sequences asdefined herein, preferably the amino acid sequences encoded by theinventive nucleic acid sequence as defined herein or the amino acidsequences themselves, the sequences can be aligned in order to besubsequently compared to one another. Therefore, e.g. a position of afirst sequence may be compared with the corresponding position of thesecond sequence. If a position in the first sequence is occupied by thesame component as is the case at a position in the second sequence, thetwo sequences are identical at this position. If this is not the case,the sequences differ at this position. If insertions occur in the secondsequence in comparison to the first sequence, gaps can be inserted intothe first sequence to allow a further alignment. If deletions occur inthe second sequence in comparison to the first sequence, gaps can beinserted into the second sequence to allow a further alignment. Thepercentage to which two sequences are identical is then a function ofthe number of identical positions divided by the total number ofpositions including those positions which are only occupied in onesequence. The percentage to which two sequences are identical can bedetermined using a mathematical algorithm. A preferred, but notlimiting, example of a mathematical algorithm which can be used is thealgorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul etal. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm isintegrated in the BLAST program. Sequences which are identical to thesequences of the present invention to a certain extent can be identifiedby this program.

The inventive nucleic acid sequence as defined herein may encodederivatives of a peptide or protein. Such a derivative of a peptide orprotein is a molecule that is derived from another molecule, such assaid peptide or protein. A “derivative” typically contains thefull-length sequence of the natural peptide or protein and additionalsequence features, e.g. at the N- or at the C-terminus, which mayexhibit an additional function to the natural full-lengthpeptide/protein. Again such derivatives have the same biologicalfunction or specific activity or at least retain an activity of thenatural full length protein of at least 50%, more preferably at least70%, even more preferably at least 90% (measured in an appropriatefunctional assay, see above, e.g. as expressed by cytokine expressionand/or secretion in an immunological reaction) as compared to thefull-length native peptide or protein, e.g. its specific therapeuticproperty. Thereby, a “derivative” of a peptide or protein alsoencompasses (chimeric) fusion peptides/proteins comprising a peptide orprotein used in the present invention or a natural full-length protein(or variant/fragment thereof) fused to a distinct peptide/proteinawarding e.g. two or more biological functions to the fusionpeptide/protein. For example, the fusion comprises a label, such as, forexample, an epitope, e.g., a FLAG epitope or a V5 epitope or an HAepitope. For example, the epitope is a FLAG epitope. Such a tag isuseful for, for example, purifying the fusion protein.

In this context, a “variant” of a protein or peptide may have at least70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over astretch of 10, 20, 30, 50, 75 or 100 amino acids of such protein orpeptide. Analogously, a “variant” of a nucleic acid sequence, orparticularly, a fragment, may have at least 70%, 75%, 80%, 85%, 90%,95%, 98% or 99% nucleotide identity over a stretch of 10, 20, 30, 50, 75or 100 nucleotide of such nucleic acid sequence; typically, however,referring to the naturally occurring full-length sequences. In case of“fragments” typically, sequence identity is determined for the fragmentover length (of the fragment) on the portion of the full-length protein(reflecting the same length as the fragment), which exhibits the highestlevel of sequence identity.

In a further preferred embodiment of the first aspect of the presentinvention the inventive nucleic acid sequence is associated with avehicle, transfection or complexation agent for increasing thetransfection efficiency and/or the immunostimulatory properties of theinventive nucleic acid sequence. Particularly preferred agents in thiscontext suitable for increasing the transfection efficiency are cationicor polycationic compounds, including protamine, nucleoline, spermine orspermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSVVP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs),PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s),Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, pIsl, FGF, Lactoferrin, Transportan, Buforin-2,Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, or histones.Additionally, preferred cationic or polycationic proteins or peptidesmay be selected from the following proteins or peptides having thefollowing total formula:(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), whereinl+m+n+o+x=8-15, and l, m, n or o independently of each other may be anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide.Particularly preferred cationic peptides in this context are e.g. Arg₇,Arg₈, Arg₉, H₃R₉, R₉H₃, H₃R₉H₃, YSSR₉SSY, (RKH)₄, Y(RKH)₂R, etc. Furtherpreferred cationic or polycationic compounds, which can be used astransfection agent may include cationic polysaccharides, for examplechitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI),cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g polyethyleneglycole); etc.

In this context it is particularly preferred that the inventive nucleicacid is complexed at least partially with a cationic or polycationiccompound, preferably cationic proteins or peptides. Partially means thatonly a part of the inventive nucleic acid is complexed with a cationicor polycationic compound and that the rest of the inventive nucleic acidis in uncomplexed form (“free”). Preferably the ratio of complexednucleic acid to: free nucleic acid is selected from a range of about 5:1(w/w) to about 1:10 (w/w), more preferably from a range of about 4:1(w/w) to about 1:8 (w/w), even more preferably from a range of about 3:1(w/w) to about 1:5 (w/w) or 1:3 (w/w), and most preferably the ratio ofcomplexed nucleic acid to free nucleic acid is selected from a ratio ofabout 1:1 (w/w).

It is preferred that the nucleic acid sequence of the invention isprovided in either naked form or complexed, e.g. by polycationiccompounds of whatever chemical structure, preferably polycationic(poly)peptides or synthetic polycationic compounds. Preferably, thenucleic acid sequence is not provided together with a packaging cell.

In a further aspect the invention provides for a composition or kit orkit of parts comprising a plurality or more than one, preferably 2 to10, more preferably 2 to 5, most preferably 2 to 4 of the inventivenucleic acid sequences as defined herein. These inventive compositionscomprise more than one inventive nucleic acid sequences, preferablyencoding different peptides or proteins which comprise preferablydifferent tumour antigens or fragments, variants or derivatives thereof.

In a preferred embodiment the inventive composition or kit or kit ofparts comprising a plurality (which means typically more than 1, 2, 3,4, 5, 6 or more than 10 nucleic acids, e.g. 2 to 10, preferably 2 to 5nucleic acids) of inventive nucleic acid sequences, particularly for usein the treatment of prostate cancer (PCa) comprises at least:

-   -   a) an inventive nucleic acid encoding at least one peptide or        protein, wherein said encoded peptide or protein comprises the        tumour antigen PSA, or a fragment, variant or derivative        thereof; and    -   b) an inventive nucleic acid encoding at least one peptide or        protein, wherein said encoded peptide or protein comprises the        tumour antigen PSMA, or a fragment, variant or derivative        thereof; and    -   c) an inventive nucleic acid encoding at least one peptide or        protein, wherein said encoded peptide or protein comprises the        tumour antigen PSCA, or a fragment, variant or derivative        thereof; and    -   d) an inventive nucleic acid encoding at least one peptide or        protein, wherein said encoded peptide or protein comprises the        tumour antigen STEAP-1, or a fragment, variant or derivative        thereof.

In a further preferred embodiment the inventive composition or kit orkit of parts comprising a plurality (which means typically more than 1,2, 3, 4, 5, 6 or more than 10 nucleic acids, e.g. 2 to 10, preferably 2to 5 nucleic acids) of inventive nucleic acid sequences, particularlyfor use in the treatment of non-small lung cancer (NSCLC) comprises atleast:

-   -   a) a nucleic acid sequence comprising or coding for        -   i. a coding region, encoding at least one peptide or protein            which comprises the tumour antigen NY-ESO-1, or a fragment,            variant or derivative thereof,        -   ii. at least one histone stem-loop, and        -   iii. a poly(A) sequence or a polyadenylation signal;    -   b) an inventive nucleic acid encoding at least one peptide or        protein, wherein said encoded peptide or protein comprises the        tumour antigen 5T4, or a fragment, variant or derivative        thereof; and    -   c) an inventive nucleic acid encoding at least one peptide or        protein, wherein said encoded peptide or protein comprises the        tumour antigen Survivin, or a fragment, variant or derivative        thereof.

Furthermore in an alternative, the inventive composition or kit or kitof parts comprising a plurality (which means typically more than 1, 2,3, 4, 5, 6 or more than 10 nucleic acids, e.g. 2 to 10, preferably 2 to5 nucleic acids) of inventive nucleic acid sequences, particularly foruse in the treatment of non-small lung cancer (NSCLC) comprises atleast:

-   -   a) a nucleic acid sequence comprising or coding for        -   i. a coding region, encoding at least one peptide or protein            which comprises the tumour antigen NY-ESO-1, or a fragment,            variant or derivative thereof,        -   ii. at least one histone stem-loop, and        -   iii. a poly(A) sequence or a polyadenylation signal;    -   b) an inventive nucleic acid encoding at least one peptide or        protein, wherein said encoded peptide or protein comprises the        tumour antigen 5T4, or a fragment, variant or derivative        thereof; and    -   c) an inventive nucleic acid encoding at least one peptide or        protein, wherein said encoded peptide or protein comprises the        tumour antigen Survivin, or a fragment, variant or derivative        thereof; and    -   d) an inventive nucleic acid encoding at least one peptide or        protein, wherein said encoded peptide or protein comprises the        tumour antigen MAGE-C1, or a fragment, variant or derivative        thereof; and    -   e) an inventive nucleic acid encoding at least one peptide or        protein, wherein said encoded peptide or protein comprises the        tumour antigen MAGE-C2, or a fragment, variant or derivative        thereof.

According to a further aspect, the present invention also provides amethod for increasing the expression of an encoded peptide or proteincomprising the steps, e.g. a) providing the inventive nucleic acidsequence as defined herein or the inventive composition comprising aplurality (which means typically more than 1, 2, 3, 4, 5, 6 or more than10 nucleic acids, e.g. 2 to 10, preferably 2 to 5 nucleic acids) ofinventive nucleic acid sequences as defined herein, b) applying oradministering the inventive nucleic acid sequence as defined herein orthe inventive composition comprising a plurality of inventive nucleicacid sequences as defined herein to an expression system, e.g. to acell-free expression system, a cell (e.g. an expression host cell or asomatic cell), a tissue or an organism. The method may be applied forlaboratory, for research, for diagnostic, for commercial production ofpeptides or proteins and/or for therapeutic purposes. In this context,typically after preparing the inventive nucleic acid sequence as definedherein or of the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein, it is typicallyapplied or administered to a cell-free expression system, a cell (e.g.an expression host cell or a somatic cell), a tissue or an organism,e.g. in naked or complexed form or as a pharmaceutical composition orvaccine as described herein, preferably via transfection or by using anyof the administration modes as described herein. The method may becarried out in vitro, in vivo or ea vivo. The method may furthermore becarried out in the context of the treatment of a specific disease,particularly in the treatment of cancer or tumour diseases, preferablyas defined herein.

In this context in vitro is defined herein as transfection ortransduction of the inventive nucleic acid as defined herein or of theinventive composition comprising a plurality of inventive nucleic acidsequences as defined herein into cells in culture outside of anorganism; in vivo is defined herein as transfection or transduction ofthe inventive nucleic acid or of the inventive composition comprising aplurality of inventive nucleic acid sequences into cells by applicationof the inventive nucleic acid or of the inventive composition to thewhole organism or individual and ea vivo is defined herein astransfection or transduction of the inventive nucleic acid or of theinventive composition comprising a plurality of inventive nucleic acidsequences (which means typically more than 1, 2, 3, 4, 5, 6 or more than10 nucleic acids, e.g. 2 to 10, preferably 2 to 5 nucleic acids) intocells outside of an organism or individual and subsequent application ofthe transfected cells to the organism or individual.

Likewise, according to another aspect, the present invention alsoprovides the use of the inventive nucleic acid sequence as definedherein or of the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein, preferably fordiagnostic or therapeutic purposes, for increasing the expression of anencoded peptide or protein, e.g. by applying or administering theinventive nucleic acid sequence as defined herein or of the inventivecomposition comprising a plurality of inventive nucleic acid sequencesas defined herein, e.g. to a cell-free expression system, a cell (e.g.an expression host cell or a somatic cell), a tissue or an organism. Theuse may be applied for laboratory, for research, for diagnostic forcommercial production of peptides or proteins and/or for therapeuticpurposes. In this context, typically after preparing the inventivenucleic acid sequence as defined herein or of the inventive compositioncomprising a plurality of inventive nucleic acid sequences as definedherein, it is typically applied or administered to a cell-freeexpression system, a cell (e.g. an expression host cell or a somaticcell), a tissue or an organism, preferably in naked form or complexedform, or as a pharmaceutical composition or vaccine as described herein,preferably via transfection or by using any of the administration modesas described herein. The use may be carried out in vitro, in vivo or exvivo. The use may furthermore be carried out in the context of thetreatment of a specific disease, particularly in the treatment of canceror tumour diseases, preferably as defined herein.

In yet another aspect the present invention also relates to an inventiveexpression system comprising an inventive nucleic acid sequence orexpression vector or plasmid according to the first aspect of thepresent invention. In this context the expression system may be acell-free expression system (e.g. an in vitro transcription/translationsystem), a cellular expression system (e.g. mammalian cells like CHOcells, insect cells, yeast cells, bacterial cells like E. coli) ororganisms used for expression of peptides or proteins (e.g. plants oranimals like cows).

Additionally, according to another aspect, the present invention alsorelates to the use of the inventive nucleic acid as defined herein or ofthe inventive composition comprising a plurality of inventive nucleicacid sequences as defined herein for the preparation of a pharmaceuticalcomposition for increasing the expression of an encoded peptide orprotein, e.g. for treating a cancer or tumour disease, preferably asdefined herein, e.g. applying or administering the inventive nucleicacid as defined herein or of the inventive composition comprising aplurality of inventive nucleic acid sequences as defined herein to acell (e.g. an expression host cell or a somatic cell), a tissue or anorganism, preferably in naked form or complexed form or as apharmaceutical composition or vaccine as described herein, morepreferably using any of the administration modes as described herein.

Accordingly, in a particular preferred aspect, the present inventionalso provides a pharmaceutical composition, comprising an inventivenucleic acid as defined herein or an inventive composition comprising aplurality of inventive nucleic acid sequences as defined herein andoptionally a pharmaceutically acceptable carrier and/or vehicle.

As a first ingredient, the inventive pharmaceutical compositioncomprises at least one inventive nucleic acid as defined herein.

As a second ingredient the inventive pharmaceutical composition mayoptional comprise at least one additional pharmaceutically activecomponent. A pharmaceutically active component in this connection is acompound that has a therapeutic effect to heal, ameliorate or prevent aparticular indication or disease as mentioned herein, preferably canceror tumour diseases. Such compounds include, without implying anylimitation, peptides or proteins, preferably as defined herein, nucleicacids, preferably as defined herein, (therapeutically active) lowmolecular weight organic or inorganic compounds (molecular weight lessthan 5000, preferably less than 1000), sugars, antigens or antibodies,preferably as defined herein, therapeutic agents already known in theprior art, antigenic cells, antigenic cellular fragments, cellularfractions; cell wall components (e.g. polysaccharides), modified,attenuated or de-activated (e.g. chemically or by irradiation) pathogens(virus, bacteria etc.), adjuvants, preferably as defined herein, etc.

Furthermore, the inventive pharmaceutical composition may comprise apharmaceutically acceptable carrier and/or vehicle. In the context ofthe present invention, a pharmaceutically acceptable carrier typicallyincludes the liquid or non-liquid basis of the inventive pharmaceuticalcomposition. If the inventive pharmaceutical composition is provided inliquid form, the carrier will typically be pyrogen-free water; isotonicsaline or buffered (aqueous) solutions, e.g phosphate, citrate etc.buffered solutions. The injection buffer may be hypertonic, isotonic orhypotonic with reference to the specific reference medium, i.e. thebuffer may have a higher, identical or lower salt content with referenceto the specific reference medium, wherein preferably such concentrationsof the afore mentioned salts may be used, which do not lead to damage ofcells due to osmosis or other concentration effects. Reference media aree.g. liquids occurring in “in vivo” methods, such as blood, lymph,cytosolic liquids, or other body liquids, or e.g. liquids, which may beused as reference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled person.Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well for the inventivepharmaceutical composition, which are suitable for administration to apatient to be treated. The term “compatible” as used here means thatthese constituents of the inventive pharmaceutical composition arecapable of being mixed with the inventive nucleic acid as defined hereinin such a manner that no interaction occurs which would substantiallyreduce the pharmaceutical effectiveness of the inventive pharmaceuticalcomposition under typical use conditions.

According to a specific embodiment, the inventive pharmaceuticalcomposition may comprise an adjuvant. In this context, an adjuvant maybe understood as any compound, which is suitable to initiate or increasean immune response of the innate immune system, i.e. a non-specificimmune response. With other words, when administered, the inventivepharmaceutical composition preferably elicits an innate immune responsedue to the adjuvant, optionally contained therein. Preferably, such anadjuvant may be selected from an adjuvant known to a skilled person andsuitable for the present case, i.e. supporting the induction of aninnate immune response in a mammal, e.g. an adjuvant protein as definedabove or an adjuvant as defined in the following.

Particularly preferred as adjuvants suitable for depot and delivery arecationic or polycationic compounds as defined above for the inventivenucleic acid sequence as vehicle, transfection or complexation agent.

The inventive pharmaceutical composition can additionally contain one ormore auxiliary substances in order to increase its immunogenicity orimmunostimulatory capacity, if desired. A synergistic action of theinventive nucleic acid sequence as defined herein and of an auxiliarysubstance, which may be optionally contained in the inventivepharmaceutical composition, is preferably achieved thereby. Depending onthe various types of auxiliary substances, various mechanisms can comeinto consideration in this respect. For example, compounds that permitthe maturation of dendritic cells (DCs), for examplelipopolysaccharides, TNF-alpha or CD40 ligand, form a first class ofsuitable auxiliary substances. In general, it is possible to use asauxiliary substance any agent that influences the immune system in themanner of a “danger signal” (LPS, GP96, etc.) or cytokines, such asGM-CFS, which allow an immune response to be enhanced and/or influencedin a targeted manner. Particularly preferred auxiliary substances arecytokines, such as monokines, lymphokines, interleukins or chemokines,that further promote the innate immune response, such as IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24,IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33,IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta orTNF-alpha, growth factors, such as hGH.

Further additives which may be included in the inventive pharmaceuticalcomposition are emulsifiers, such as, for example, TWEEN®, non-ionicdetergent; wetting agents, such as, for example, sodium lauryl sulfate;colouring agents; taste-imparting agents, pharmaceutical carriers;tablet-forming agents; stabilizers; antioxidants; preservatives.

The inventive pharmaceutical composition can also additionally containany further compound, which is known to be immunostimulating due to itsbinding affinity (as ligands) to human Toll-like receptors TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

The inventive pharmaceutical composition may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulmonal, intraperitoneal, intracardial, intraarterial, andsublingual injection or infusion techniques.

Preferably, the inventive pharmaceutical composition may be administeredby parenteral injection, more preferably by subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial, transdermal,intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, and sublingual injection or via infusion techniques.Particularly preferred is intradermal and intramuscular injection.Sterile injectable forms of the inventive pharmaceutical compositionsmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents.

The inventive pharmaceutical composition as defined herein may also beadministered orally in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions.

The inventive pharmaceutical composition may also be administeredtopically, especially when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be formulated in a suitable ointment, containing theinventive nucleic acid as defined herein suspended or dissolved in oneor more carriers.

The inventive pharmaceutical composition typically comprises a “safe andeffective amount” of the components of the inventive pharmaceuticalcomposition, particularly of the inventive nucleic acid sequence(s) asdefined herein. As used herein, a “safe and effective amount” means anamount of the inventive nucleic acid sequence(s) as defined herein assuch that is sufficient to significantly induce a positive modificationof a disease or disorder as defined herein. At the same time, however, a“safe and effective amount” is small enough to avoid seriousside-effects and to permit a sensible relationship between advantage andrisk. The determination of these limits typically lies within the scopeof sensible medical judgment.

The inventive pharmaceutical composition may be used for human and alsofor veterinary medical purposes, preferably for human medical purposes,as a pharmaceutical composition in general or as a vaccine.

According to another particularly preferred aspect, the inventivepharmaceutical composition (or the inventive nucleic acid sequence asdefined herein or the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein) may be provided orused as a vaccine. Typically, such a vaccine is as defined above forpharmaceutical compositions. Additionally, such a vaccine typicallycontains the inventive nucleic acid as defined herein or the inventivecomposition comprising a plurality of inventive nucleic acid sequencesas defined herein.

The inventive vaccine may also comprise a pharmaceutically acceptablecarrier, adjuvant, and/or vehicle as defined herein for the inventivepharmaceutical composition. In the specific context of the inventivevaccine, the choice of a pharmaceutically acceptable carrier isdetermined in principle by the manner in which the inventive vaccine isadministered. The inventive vaccine can be administered, for example,systemically or locally. Routes for systemic administration in generalinclude, for example, transdermal, oral, parenteral routes, includingsubcutaneous, intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,vaccines may be administered by an intradermal, subcutaneous, orintramuscular route. Inventive vaccines are therefore preferablyformulated in liquid (or sometimes in solid) form.

The inventive vaccine can additionally contain one or more auxiliarysubstances in order to increase its immunogenicity or immunostimulatorycapacity, if desired. Particularly preferred are adjuvants as auxiliarysubstances or additives as defined for the pharmaceutical composition.

The present invention furthermore provides several applications and usesof the inventive nucleic acid sequence as defined herein, of theinventive composition comprising a plurality of inventive nucleic acidsequences as defined herein, of the inventive pharmaceuticalcomposition, of the inventive vaccine, all comprising the inventivenucleic acid sequence as defined herein or of kits comprising same.

According to one specific aspect, the present invention is directed tothe first medical use of the inventive nucleic acid sequence as definedherein or of the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein as a medicament,preferably as a vaccine particularly in the treatment of cancer ortumour diseases.

According to another aspect, the present invention is directed to thesecond medical use of the inventive nucleic acid sequence as definedherein or of the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein, for the treatment ofcancer and tumour diseases as defined herein, preferably to the use ofthe inventive nucleic acid sequence as defined herein, of the inventivecomposition comprising a plurality of inventive nucleic acid sequencesas defined herein, of a pharmaceutical composition or vaccine comprisingsame or of kits comprising same for the preparation of a medicament forthe prophylaxis, treatment and/or amelioration of cancer or tumourdiseases as defined herein. Preferably, the pharmaceutical compositionor a vaccine is used or to be administered to a patient in need thereoffor this purpose.

Preferably, diseases as mentioned herein are selected from cancer ortumour diseases which preferably include e.g. Acute lymphoblasticleukemia, Acute myeloid leukemia, Adrenocortical carcinoma, AIDS-relatedcancers, AIDS-related lymphoma, Anal cancer, Appendix cancer,Astrocytoma, Basal cell carcinoma, Bile duct cancer, Bladder cancer,Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma, Brainstemglioma, Brain tumor, cerebellar astrocytoma, cerebralastrocytoma/malignant glioma, ependymoma, medulloblastoma,supratentorial primitive neuroectodermal tumors, visual pathway andhypothalamic glioma, Breast cancer, Bronchial adenomas/carcinoids,Burkitt lymphoma, childhood Carcinoid tumor, gastrointestinal Carcinoidtumor, Carcinoma of unknown primary, primary Central nervous systemlymphoma, childhood Cerebellar astrocytoma, childhood Cerebralastrocytoma/Malignant glioma, Cervical cancer, Childhood cancers,Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Chronicmyeloproliferative disorders, Colon Cancer, Cutaneous T-cell lymphoma,Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma,Esophageal cancer, Ewing's sarcoma in the Ewing family of tumors,Childhood Extracranial germ cell tumor, Extragonadal Germ cell tumor,Extrahepatic bile duct cancer, Intraocular melanoma, Retinoblastoma,Gallbladder cancer, Gastric (Stomach) cancer, Gastrointestinal CarcinoidTumor, Gastrointestinal stromal tumor (GIST), extracranial,extragonadal, or ovarian Germ cell tumor, Gestational trophoblastictumor, Glioma of the brain stem, Childhood Cerebral Astrocytoma,Childhood Visual Pathway and Hypothalamic Glioma, Gastric carcinoid,Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular(liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, childhoodHypothalamic and visual pathway glioma, Intraocular Melanoma, Islet CellCarcinoma (Endocrine Pancreas), Kaposi sarcoma, Kidney cancer (renalcell cancer), Laryngeal Cancer, Leukemias, acute lymphoblastic Leukemia,acute myeloid Leukemia, chronic lymphocytic Leukemia, chronicmyelogenous Leukemia, hairy cell Leukemia, Lip and Oral Cavity Cancer,Liposarcoma, Liver Cancer, Non-Small Cell Lung Cancer, Small Cell LungCancer, Lymphomas, AIDS-related Lymphoma, Burkitt Lymphoma, cutaneousT-Cell Lymphoma, Hodgkin Lymphoma, Non-Hodgkin Lymphomas, PrimaryCentral Nervous System Lymphoma, Waldenström Macroglobulinemia,Malignant Fibrous Histiocytoma of Bone/Osteosarcoma, ChildhoodMedulloblastoma, Melanoma, Intraocular (Eye) Melanoma, Merkel CellCarcinoma, Adult Malignant Mesothelioma, Childhood Mesothelioma,Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer,Childhood Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/PlasmaCell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Diseases, Chronic MyelogenousLeukemia, Adult Acute Myeloid Leukemia, Childhood Acute MyeloidLeukemia, Multiple Myeloma (Cancer of the Bone-Marrow), ChronicMyeloproliferative Disorders, Nasal cavity and paranasal sinus cancer,Nasopharyngeal carcinoma, Neuroblastoma, Oral Cancer, Oropharyngealcancer, Osteosarcoma/malignant fibrous histiocytoma of bone, Ovariancancer, Ovarian epithelial cancer (Surface epithelial-stromal tumor),Ovarian germ cell tumor, Ovarian low malignant potential tumor,Pancreatic cancer, islet cell Pancreatic cancer, Paranasal sinus andnasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngealcancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma,childhood Pineoblastoma and supratentorial primitive neuroectodermaltumors, Pituitary adenoma, Plasma cell neoplasia/Multiple myeloma,Pleuropulmonary blastoma, Primary central nervous system lymphoma,Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer),Cancer of the Renal pelvis and ureter, Retinoblastoma, childhoodRhabdomyosarcoma, Salivary gland cancer, Sarcoma of the Ewing family oftumors, Kaposi Sarcoma, soft tissue Sarcoma, uterine Sarcoma, Sézarysyndrome, Skin cancer (nonmelanoma), Skin cancer (melanoma), Merkel cellSkin carcinoma, Small intestine cancer, Squamous cell carcinoma,metastatic Squamous neck cancer with occult primary, childhoodSupratentorial primitive neuroectodermal tumor, Testicular cancer,Throat cancer, childhood Thymoma, Thymoma and Thymic carcinoma, Thyroidcancer, childhood Thyroid cancer, Transitional cell cancer of the renalpelvis and ureter, gestational Trophoblastic tumor, Urethral cancer,endometrial Uterine cancer, Uterine sarcoma, Vaginal cancer, childhoodVisual pathway and hypothalamic glioma, Vulvar cancer, Waldenströmmacroglobulinemia, and childhood Wilms tumor (kidney cancer).

In a further preferred aspect, the inventive nucleic acid sequence asdefined herein or the inventive composition comprising a plurality ofinventive nucleic acid sequences as defined herein may be used for thepreparation of a pharmaceutical composition or a vaccine, particularlyfor purposes as defined herein.

The inventive pharmaceutical composition or vaccine may furthermore beused for the treatment of a disease or a disorder, preferably of canceror tumour diseases as defined herein.

According to a final aspect, the present invention also provides kits,particularly kits of parts. Such kits, particularly kits of parts,typically comprise as components alone or in combination with furthercomponents as defined herein at least one inventive nucleic acidsequence as defined herein, the inventive pharmaceutical composition orvaccine comprising the inventive nucleic acid sequence. The at least oneinventive nucleic acid sequence as defined herein, is optionally incombination with further components as defined herein, whereby the atleast one nucleic acid of the invention is provided separately (firstpart of the kit) from at least one other part of the kit comprising oneor more other components. The inventive pharmaceutical compositionand/or the inventive vaccine may e.g. occur in one or different parts ofthe kit. As an example, e.g. at least one part of the kit may compriseat least one inventive nucleic acid sequence as defined herein, and atleast one further part of the kit at least one other component asdefined herein, e.g. at least one other part of the kit may comprise atleast one pharmaceutical composition or vaccine or a part thereof, e.g.at least one part of the kit may comprise the inventive nucleic acidsequence as defined herein, at least one further part of the kit atleast one other component as defined herein, at least one further partof the kit at least one component of the inventive pharmaceuticalcomposition or vaccine or the inventive pharmaceutical composition orvaccine as a whole, and at least one further part of the kit e.g. atleast one pharmaceutical carrier or vehicle, etc. In case the kit or kitof parts comprises a plurality of inventive nucleic acid sequences, onecomponent of the kit can comprise only one, several or all inventivenucleic acid sequences comprised in the kit. In an alternativeembodiment each/every inventive nucleic acid sequence may be comprisedin a different/separate component of the kit such that each componentforms a part of the kit. Also, more than one nucleic acid may becomprised in a first component as part of the kit, whereas one or moreother (second, third etc.) components (providing one or more other partsof the kit) may either contain one or more than one inventive nucleicacids, which may be identical or partially identical or different fromthe first component. The kit or kit of parts may furthermore containtechnical instructions with information on the administration and dosageof the inventive nucleic acid sequence, the inventive pharmaceuticalcomposition or the inventive vaccine or of any of its components orparts, e.g. if the kit is prepared as a kit of parts.

Taken together, the invention provides a nucleic acid sequencecomprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;        wherein said peptide or protein comprises a tumour antigen a        fragment, variant or derivative of said tumour antigen,        preferably, wherein the tumour antigen is a melanocyte-specific        antigen, a cancer-testis antigen or a tumour-specific antigen,        preferably a CT-X antigen, a non-X CT-antigen, a binding partner        for a CT-X antigen or a binding partner for a non-X CT-antigen        or a tumour-specific antigen, more preferably a CT-X antigen, a        binding partner for a non-X CT-antigen or a tumour-specific        antigen or a fragment, variant or derivative of said tumour        antigen.

In a further preferred embodiment, the invention relates to acomposition comprising at least one type of nucleic acid sequencecomprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;    -   wherein said peptide or protein comprises a tumour antigen a        fragment, variant or derivative of said tumour antigen,        preferably, wherein the tumour antigen is a melanocyte-specific        antigen, a cancer-testis antigen or a tumour-specific antigen,        preferably a CT-X antigen, a non-X CT-antigen, a binding partner        for a CT-X antigen or a binding partner for a non-X CT-antigen        or a tumour-specific antigen, more preferably a CT-X antigen, a        binding partner for a non-X CT-antigen or a tumour-specific        antigen or a fragment, variant or derivative of said tumour        antigen; and wherein each of the nucleic acid sequences encodes        a different peptide or protein.

The composition may comprise further an pharmaceutically acceptablecarrier and/or pharmaceutically acceptable adjuvants as defined herein.The composition may be used as a vaccine or for treatment of a diseaseassociated with cancer or tumour.

In a further preferred embodiment, the invention provides a compositioncomprising at least two, preferably two or more, more preferably aplurality of nucleic acid sequences sequence (which means typically morethan 1, 2, 3, 4, 5, 6 or more than 10 nucleic acids, e.g. 2 to 10,preferably 2 to 5 nucleic acids) comprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;        -   wherein said peptide or protein comprises a tumour antigen a            fragment, variant or derivative of said tumour antigen,            preferably, wherein the tumour antigen is a            melanocyte-specific antigen, a cancer-testis antigen or a            tumour-specific antigen, preferably a CT-X antigen, a non-X            CT-antigen, a binding partner for a CT-X antigen or a            binding partner for a non-X CT-antigen or a tumour-specific            antigen, more preferably a CT-X antigen, a binding partner            for a non-X CT-antigen or a tumour-specific antigen or a            fragment, variant or derivative of said tumour antigen; and            wherein each of the nucleic acid sequences encodes a            different peptide or protein.

The composition may comprise further an pharmaceutically acceptablecarrier and/or pharmaceutically acceptable adjuvants as defined herein.The composition may be used as a vaccine or for treatment of a diseaseassociated with cancer or tumour.

In a further preferred embodiment, the invention provides a compositioncomprising at least two, preferably two or more, more preferably aplurality (which means typically more than 1, 2, 3, 4, 5, 6 or more than10 nucleic acids, e.g. 2 to 10, preferably 2 to 5 nucleic acids) ofnucleic acid sequences sequence comprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;        -   wherein said peptide or protein comprises a tumour antigen a            fragment, variant or derivative of said tumour antigen,            preferably, wherein the tumour antigen is a            melanocyte-specific antigen, a cancer-testis antigen or a            tumour-specific antigen, preferably a CT-X antigen, a non-X            CT-antigen, a binding partner for a CT-X antigen or a            binding partner for a non-X CT-antigen or a tumour-specific            antigen, more preferably a CT-X antigen, a binding partner            for a non-X CT-antigen or a tumour-specific antigen or a            fragment, variant or derivative of said tumour antigen; and            wherein each of the nucleic acid sequences encodes a            different peptide or protein; and preferably wherein each            type of nucleic acid sequence encodes for a different            peptide or protein, preferably for a different tumour            antigen.

The composition may comprise further an pharmaceutically acceptablecarrier and/or pharmaceutically acceptable adjuvants as defined herein.The composition may be used as a vaccine or for treatment of a diseaseassociated with cancer or tumour.

In a further preferred embodiment, the invention provides a compositioncomprising at least two, preferably two or more, more preferably aplurality of nucleic acid sequences sequence (which means typically morethan 1, 2, 3, 4, 5, 6 or more than 10 nucleic acids, e.g. 2 to 10,preferably 2 to 5 nucleic acids) comprising or coding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;        -   wherein said peptide or protein comprises a tumour antigen a            fragment, variant or derivative of said tumour antigen,            preferably, wherein the tumour antigen is a            melanocyte-specific antigen, a cancer-testis antigen or a            tumour-specific antigen, preferably a CT-X antigen, a non-X            CT-antigen, a binding partner for a CT-X antigen or a            binding partner for a non-X CT-antigen or a tumour-specific            antigen, more preferably a CT-X antigen, a binding partner            for a non-X CT-antigen or a tumour-specific antigen or a            fragment, variant or derivative of said tumour antigen; and            wherein each of the nucleic acid sequences encodes a            different peptide or protein; and preferably wherein each            type of nucleic acid sequence encodes for a different            peptide or protein, preferably for a different tumour            antigen, more preferably, wherein one type of the contained            nucleic acid sequences encodes for PSA, PSMA, PSCA, STEAP-1,            NY-ESO-1, 5T4, Survivin, MAGE-C1, or MAGE-C2.

The composition may comprise further an pharmaceutically acceptablecarrier and/or pharmaceutically acceptable adjuvants as defined herein.The composition may be used as a vaccine or for treatment of a diseaseassociated with cancer or tumour.

In some embodiments, it may be preferred, provided that the compositioncontains only one type of nucleic acid sequence, if the nucleic acidsequence does not encode for NY-ESO1, provided that the compositioncontains only one type of nucleic acid sequence.

In a further preferred embodiment, the invention provides a compositioncomprising at least two nucleic acid sequences sequence comprising orcoding for

-   -   a) a coding region, encoding at least one peptide or protein;    -   b) at least one histone stem-loop, and    -   c) a poly(A) sequence or a polyadenylation signal;        -   wherein said peptide or protein comprises a tumour antigen a            fragment, variant or derivative of said tumour antigen,            preferably, wherein the tumour antigen is a            melanocyte-specific antigen, a cancer-testis antigen or a            tumour-specific antigen, preferably a CT-X antigen, a non-X            CT-antigen, a binding partner for a CT-X antigen or a            binding partner for a non-X CT-antigen or a tumour-specific            antigen, more preferably a CT-X antigen, a binding partner            for a non-X CT-antigen or a tumour-specific antigen or a            fragment, variant or derivative of said tumour antigen; and            wherein each of the nucleic acid sequences encodes a            different peptide or protein; and wherein at least one of            the nucleic acid sequences encodes for 5T4, 707-AP, 9D7,            AFP, AlbZIP HPG1, alpha-5-beta-1-integrin,            alpha-5-beta-6-integrin, alpha-actinin-4/m,            alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4,            BAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m,            BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125,            calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L,            CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56,            CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66,            COA-1/m, coactosin-like protein, collage XXIII, COX-2,            CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10,            DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam,            EphA2, EphAs, ErbBs, ETV6-AML1, EZH2, FGF-5, FN, Frau-1,            G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,            GAGE7b, GAGE-s, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE,            HAST-2, hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R17I,            HLA-A11/m, HLA-A2/m, HNE, homeobox NKX3.1, HOM-TES-14/SCP-1,            HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE,            IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor,            kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,            KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2,            MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12,            MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6,            MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3,            MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1,            MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2,            MART-2/m, matrix protein 22, MC1R, M-CSF, ME1/m, mesothelin,            MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2,            MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A,            N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m,            NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-B, OA1,            OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin,            osteopontin, p15, p190 minor bcr-abl, p53, p53/m, PAGE-4,            PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1-Kinase, Pin-1,            Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein, proteinase-3,            PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,            RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3,            SCC, SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1,            STEAP-1, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90,            TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m,            TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase,            UPA, VEGFR1, VEGFR-2/FLK-1, WT1 and a immunoglobulin            idiotype of a lymphoid blood cell or a T cell receptor            idiotype of a lymphoid blood cell, or a fragment, variant or            derivative of said tumour antigen; preferably survivin or a            homologue thereof, an antigen from the MAGE-family or a            binding partner thereof or a fragment, variant or derivative            of said tumour antigen.

The composition may comprise further an pharmaceutically acceptablecarrier and/or pharmaceutically acceptable adjuvants as defined herein.The composition may be used as a vaccine or for treatment of a diseaseassociated with cancer or tumour.

In the present invention, if not otherwise indicated, different featuresof alternatives and embodiments may be combined with each other.Furthermore, the term “comprising” shall not be construed as meaning“consisting of”, if not specifically mentioned. However, in the contextof the present invention, term “comprising” may be substituted with theterm “consisting of”, where applicable.

FIGURES

The following Figures are intended to illustrate the invention furtherand shall not be construed to limit the present invention thereto.

FIG. 1: shows the histone stem-loop consensus sequence generated frommetazoan and protozoan stem loop sequences (as reported by Dávila López,M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308). 4001 histone stem-loop sequences from metazoaand protozoa were aligned and the quantity of the occurring nucleotidesis indicated for every position in the stem-loop sequence. The generatedconsensus sequence representing all nucleotides present in the sequencesanalyzed is given using the single-letter nucleotide code. In additionto the consensus sequence, sequences are shown representing at least99%, 95% and 90% of the nucleotides present in the sequences analyzed.

FIG. 2: shows the histone stem-loop consensus sequence generated fromprotozoan stem loop sequences (as reported by Dávila López, M., &Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308). 131 histone stem-loop sequences from protozoawere aligned and the quantity of the occurring nucleotides is indicatedfor every position in the stem-loop sequence. The generated consensussequence representing all nucleotides present in the sequences analyzedis given using the single-letter nucleotide code. In addition to theconsensus sequence, sequences are shown representing at least 99%, 95%and 90% of the nucleotides present in the sequences analyzed.

FIG. 3: shows the histone stem-loop consensus sequence generated frommetazoan stem loop sequences (as reported by Dávila López, M., &Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308). 3870 histone stem-loop sequences from metazoawere aligned and the quantity of the occurring nucleotides is indicatedfor every position in the stem-loop sequence. The generated consensussequence representing all nucleotides present in the sequences analyzedis given using the single-letter nucleotide code. In addition to theconsensus sequence, sequences are shown representing at least 99%, 95%and 90% of the nucleotides present in the sequences analyzed.

FIG. 4: shows the histone stem-loop consensus sequence generated fromvertebrate stem loop sequences (as reported by Dávila López, M., &Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308). 1333 histone stem-loop sequences fromvertebrates were aligned and the quantity of the occurring nucleotidesis indicated for every position in the stem-loop sequence. The generatedconsensus sequence representing all nucleotides present in the sequencesanalyzed is given using the single-letter nucleotide code. In additionto the consensus sequence, sequences are shown representing at least99%, 95% and 90% of the nucleotides present in the sequences analyzed.

FIG. 5: shows the histone stem-loop consensus sequence generated fromhuman (Homo sapiens) stem loop sequences (as reported by Dávila López,M., & Samuelsson, T. (2008), RNA (New York, N.Y.), 14(1), 1-10.doi:10.1261/rna.782308). 84 histone stem-loop sequences from humans werealigned and the quantity of the occurring nucleotides is indicated forevery position in the stem-loop sequence. The generated consensussequence representing all nucleotides present in the sequences analyzedis given using the single-letter nucleotide code. In addition to theconsensus sequence, sequences are shown representing at least 99%, 95%and 90% of the nucleotides present in the sequences analyzed.

FIGS. 6 to 21: show mRNAs from in vitro transcription.

-   -   Given are the designation and the sequence of mRNAs obtained by        in vitro transcription.    -   The following abbreviations are used:    -   ppLuc (GC): GC-enriched mRNA sequence coding for Photinus        pyralis luciferase    -   ag: 3′ untranslated region (UTR) of the alpha globin gene    -   A64: poly(A)-sequence with 64 adenylates    -   A120: poly(A)-sequence with 120 adenylates    -   histoneSL: histone stem-loop    -   aCPSL: stem loop which has been selected from a library for its        specific binding of the αCP-2KL protein    -   PolioCL: 5′ clover leaf from Polio virus genomic RNA    -   G30: poly(G) sequence with 30 guanylates    -   U30: poly(U) sequence with 30 uridylates    -   SL: unspecific/artificial stem-loop    -   N32: unspecific sequence of 32 nucleotides    -   NY-ESO-1 (G/C): GC-enriched mRNA sequence coding for the human        tumour antigen NY-ESO-1    -   Survivin(G/C): GC-enriched mRNA sequence coding for the human        tumour antigen Survivin    -   MAGE-C1(G/C): GC-enriched mRNA sequence coding for the human        tumour antigen MAGE-C1    -   Within the sequences, the following elements are highlighted:        coding region (ORF) (capital letters), ag (bold), histoneSL        (underlined), further distinct sequences tested (italic).

FIG. 6: shows the mRNA sequence of ppLuc(GC)-ag (SEQ ID NO: 43).

-   -   By linearization of the original vector at the restriction site        immediately following the alpha-globin 3′-UTR (ag), mRNA is        obtained lacking a poly(A) sequence.

FIG. 7: shows the mRNA sequence of ppLuc(GC)-ag-A64 (SEQ ID NO: 44).

-   -   By linearization of the original vector at the restriction site        immediately following the A64 poly(A)-sequence, mRNA is obtained        ending with an A64 poly(A) sequence.

FIG. 8: shows the mRNA sequence of ppLuc(GC)-ag-histoneSL (SEQ ID NO:45).

-   -   The A64 poly(A) sequence was replaced by a histoneSL. The        histone stem-loop sequence used in the examples was obtained        from Cakmakci et al. (2008). Molecular and Cellular Biology,        28(3), 1182-1194.

FIG. 9: shows the mRNA sequence of ppLuc(GC)-ag-A64-histoneSL (SEQ IDNO: 46).

-   -   The histoneSL was appended 3′ of A64 poly(A).

FIG. 10: shows the mRNA sequence of ppLuc(GC)-ag-A120 (SEQ ID NO: 47).

-   -   The A64 poly(A) sequence was replaced by an A120 poly(A)        sequence.

FIG. 11: shows the mRNA sequence of ppLuc(GC)-ag-A64-ag (SEQ ID NO: 48).A second alpha-globin 3′-UTR was appended 3′ of A64 poly(A).

FIG. 12: shows the mRNA sequence of ppLuc(GC)-ag-A64-aCPSL (SEQ ID NO:49).

-   -   A stem loop was appended 3′ of A64 poly(A). The stem loop has        been selected from a library for its specific binding of the        αCP-2KL protein (Thisted et al., (2001), The Journal of        Biological Chemistry, 276(20), 17484-17496). αCP-2KL is an        isoform of αCP-2, the most strongly expressed αCP protein        (alpha-globin mRNA poly(C) binding protein) (Makeyev et al.,        (2000), Genomics, 67(3), 301-316), a group of RNA binding        proteins, which bind to the alpha-globin 3′-UTR (Chkheidze et        al., (1999), Molecular and Cellular Biology, 19(7), 4572-4581).

FIG. 13: shows the mRNA sequence of ppLuc(GC)-ag-A64-PolioCL (SEQ ID NO:50).

-   -   The 5′ clover leaf from Polio virus genomic RNA was appended 3′        of A64 poly(A).

FIG. 14: shows the mRNA sequence of ppLuc(GC)-ag-A64-G30 (SEQ ID NO: 51)

-   -   A stretch of 30 guanylates was appended 3′ of A64 poly(A).

FIG. 15: shows the mRNA sequence of ppLuc(GC)-ag-A64-U30 (SEQ ID NO: 52)

-   -   A stretch of 30 uridylates was appended 3′ of A64 poly(A).

FIG. 16: shows the mRNA sequence of ppLuc(GC)-ag-A64-SL (SEQ ID NO: 53)

-   -   A stem loop was appended 3′ of A64 poly(A). The upper part of        the stem and the loop were taken from (Babendure et al., (2006),        RNA (New York, N.Y.), 12(5), 851-861). The stem loop consists of        a 17 base pair long, CG-rich stem and a 6 base long loop.

FIG. 17: shows ppLuc(GC)-ag-A64-N32 (SEQ ID NO: 54)

-   -   By linearization of the original vector at an alternative        restriction site, mRNA is obtained with 32 additional        nucleotides following poly(A).

FIG. 18: shows the mRNA sequence of NY-ESO-1(GC)-ag-A64-C30 (SEQ ID NO:55)

FIG. 19: shows the mRNA sequence of NY-ESO-1(GC)-ag-A64-C30-histoneSL(SEQ ID NO: 56)

FIG. 20: shows the mRNA sequence of Survivin(GC)-ag-A64-C30-histoneSL(SEQ ID NO: 57)

FIG. 21: shows the mRNA sequence of MAGE-C1(GC)-ag-A64-C30-histoneSL(SEQ ID NO: 58)

FIG. 22: shows that the combination of poly(A) and histoneSL increasesprotein expression from mRNA in a synergistic manner.

-   -   The effect of poly(A) sequence, histoneSL, and the combination        of poly(A) and histoneSL on luciferase expression from mRNA was        examined. Therefore different mRNAs were electroporated into        HeLa cells. Luciferase levels were measured at 6, 24, and 48        hours after transfection. Little luciferase is expressed from        mRNA having neither poly(A) sequence nor histoneSL. Both a        poly(A) sequence or the histoneSL increase the luciferase level.        Strikingly however, the combination of poly(A) and histoneSL        further strongly increases the luciferase level, manifold above        the level observed with either of the individual elements, thus        acting synergistically. Data are graphed as mean RLU±SD        (relative light units±standard deviation) for triplicate        transfections. Specific RLU are summarized in Example 14.2.

FIG. 23: shows that the combination of poly(A) and histoneSL increasesprotein expression from mRNA irrespective of their order.

-   -   The effect of poly(A) sequence, histoneSL, the combination of        poly(A) and histoneSL, and their order on luciferase expression        from mRNA was examined. Therefore different mRNAs were        lipofected into HeLa cells. Luciferase levels were measured at        6, 24, and 48 hours after the start of transfection. Both an A64        poly(A) sequence or the histoneSL give rise to comparable        luciferase levels. Increasing the length of the poly(A) sequence        from A64 to A120 or to A300 increases the luciferase level        moderately. In contrast, the combination of poly(A) and        histoneSL increases the luciferase level much further than        lengthening of the poly(A) sequence. The combination of poly(A)        and histoneSL acts synergistically as it increases the        luciferase level manifold above the level observed with either        of the individual elements. The synergistic effect of the        combination of poly(A) and histoneSL is seen irrespective of the        order of poly(A) and histoneSL and irrespective of the length of        poly(A) with A64-histoneSL or histoneSL-A250 mRNA. Data are        graphed as mean RLU±SD for triplicate transfections. Specific        RLU are summarized in Example 14.3.

FIG. 24: shows that the rise in protein expression by the combination ofpoly(A) and histoneSL is specific.

-   -   The effect of combining poly(A) and histoneSL or poly(A) and        alternative sequences on luciferase expression from mRNA was        examined. Therefore different mRNAs were electroporated into        HeLa cells. Luciferase levels were measured at 6, 24, and 48        hours after transfection. Both a poly(A) sequence or the        histoneSL give rise to comparable luciferase levels. The        combination of poly(A) and histoneSL strongly increases the        luciferase level, manifold above the level observed with either        of the individual elements, thus acting synergistically. In        contrast, combining poly(A) with any of the other sequences is        without effect on the luciferase level compared to mRNA        containing only a poly(A) sequence. Thus, the combination of        poly(A) and histoneSL acts specifically and synergistically.        Data are graphed as mean RLU±SD for triplicate transfections.        Specific RLU are summarized in Example 14.4.

FIG. 25: shows that the combination of poly(A) and histoneSL increasesprotein expression from mRNA in a synergistic manner in vivo.

-   -   The effect of poly(A) sequence, histoneSL, and the combination        of poly(A) and histoneSL on luciferase expression from mRNA in        vivo was examined. Therefore different mRNAs were injected        intradermally into mice. Mice were sacrificed 16 hours after        injection and Luciferase levels at the injection sites were        measured. Luciferase is expressed from mRNA having either a        histoneSL or a poly(A) sequence. Strikingly however, the        combination of poly(A) and histoneSL strongly increases the        luciferase level, manifold above the level observed with either        of the individual elements, thus acting synergistically. Data        are graphed as mean RLU±SEM (relative light units±standard error        of mean). Specific RLU are summarized in Example 14.5.

FIG. 26: shows that the combination of poly(A) and histoneSL increasesNY-ESO-1 protein expression from mRNA.

-   -   The effect of poly(A) sequence and the combination of poly(A)        and histoneSL on NY-ESO-1 expression from mRNA was examined.        Therefore different mRNAs were electroporated into HeLa cells.        NY-ESO-1 levels were measured at 24 hours after transfection by        flow cytometry. NY-ESO-1 is expressed from mRNA having only a        poly(A) sequence. Strikingly however, the combination of poly(A)        and histoneSL strongly increases the NY-ESO-1 level, manifold        above the level observed with only a poly(A) sequence. Data are        graphed as counts against fluorescence intensity. Median        fluorescence intensities (MFI) are summarized in Example 14.6.

FIG. 27: shows that the combination of poly(A) and histoneSL increasesthe level of antibodies elicited by vaccination with mRNA.

-   -   The effect of poly(A) sequence and the combination of poly(A)        and histoneSL on the induction of anti NY-ESO-1 antibodies        elicited by vaccination with mRNA was examined. Therefore        C57BL/6 mice were vaccinated intradermally with different mRNAs        complexed with protamine. The level of NY-ESO-1-specific        antibodies in vaccinated and control mice was analyzed by ELISA        with serial dilutions of sera. Anti NY-ESO-1 IgG2a[b] is induced        by mRNA having only a poly(A) sequence. Strikingly however, the        combination of poly(A) and histoneSL strongly increases the anti        NY-ESO-1 IgG2a[b] level, manifold above the level observed with        only a poly(A) sequence. Data are graphed as mean endpoint        titers. Mean endpoint titers are summarized in Example 14.7.

EXAMPLES

The following Examples are intended to illustrate the invention furtherand shall not be construed to limit the present invention thereto.

1. Generation of Histone-Stem-Loop Consensus Sequences

Prior to the experiments, histone stem-loop consensus sequences weredetermined on the basis of metazoan and protozoan histone stem-loopsequences. Sequences were taken from the supplement provided by Lopez etal. (Dávila López, M., & Samuelsson, T. (2008), RNA (New York, N.Y.),14(1), 1-10. doi:10.1261/rna.782308), who identified a large number ofnatural histone stem-loop sequences by searching genomic sequences andexpressed sequence tags. First, all sequences from metazoa and protozoa(4001 sequences), or all sequences from protozoa (131 sequences) oralternatively from metazoa (3870 sequences), or from vertebrates (1333sequences) or from humans (84 sequences) were grouped and aligned. Then,the quantity of the occurring nucleotides was determined for everyposition. Based on the tables thus obtained, consensus sequences for the5 different groups of sequences were generated representing allnucleotides present in the sequences analyzed. In addition, morerestrictive consensus sequences were also obtained, increasinglyemphasizing conserved nucleotides

2. Preparation of DNA-Templates

A vector for in vitro transcription was constructed containing a T7promoter followed by a GC-enriched sequence coding for Photinus pyralisluciferase (ppLuc(GC)), the center part of the 3′ untranslated region(UTR) of alpha-globin (ag), and a poly(A) sequence. The poly(A) sequencewas immediately followed by a restriction site used for linearization ofthe vector before in vitro transcription in order to obtain mRNA endingin an A64 poly(A) sequence. mRNA obtained from this vector accordinglyby in vitro transcription is designated as “ppLuc(GC)-ag-A64”.

Linearization of this vector at alternative restriction sites before invitro transcription allowed to obtain mRNA either extended by additionalnucleotides 3′ of A64 or lacking A64. In addition, the original vectorwas modified to include alternative sequences. In summary, the followingmRNAs were obtained from these vectors by in vitro transcription (mRNAsequences are given in FIGS. 6 to 17):

(SEQ ID NO: 43) ppLuc(GC)-ag (SEQ ID NO: 44) ppLuc(GC)-ag-A64 (SEQ IDNO: 45) ppLuc(GC)-ag-histoneSL (SEQ ID NO: 46)ppLuc(GC)-ag-A64-histoneSL (SEQ ID NO: 47) ppLuc(GC)-ag-A120 (SEQ ID NO:48) ppLuc(GC)-ag-A64-ag (SEQ ID NO: 49) ppLuc(GC)-ag-A64-aCPSL (SEQ IDNO: 50) ppLuc(GC)-ag-A64-PolioCL (SEQ ID NO: 51) ppLuc(GC)-ag-A64-G30(SEQ ID NO: 52) ppLuc(GC)-ag-A64-U30 (SEQ ID NO: 53) ppLuc(GC)-ag-A64-SL(SEQ ID NO: 54) ppLuc(GC)-ag-A64-N32

Furthermore DNA plasmid sequences coding for the tumour antigensNY-ESO-1, Survivin and MAGE-C1 were prepared accordingly as describedabove.

In summary, the following mRNAs were obtained from these vectors by invitro transcription (mRNA sequences are given in FIGS. 18 to 21):

(SEQ ID NO: 55) NY-ESO-1(GC)-ag-A62-C30 (SEQ ID NO: 56)NY-ESO-1(GC)-ag-A62-C30-histoneSL (SEQ ID NO: 57)Survivin(GC)-ag-A62-C30-histoneSL (SEQ ID NO: 58)MAGE-C1(GC)-ag-A64-C30-histoneSL

3. In Vitro Transcription

The DNA-template according to Example 2 was linearized and transcribedin vitro using T7-Polymerase. The DNA-template was then digested byDNase-treatment. All mRNA-transcripts contained a 5′-CAP structureobtained by adding an excess ofN7-Methyl-Guanosine-5′-Triphosphate-5′-Guanosine to the transcriptionreaction. mRNA thus obtained was purified and resuspended in water.

4. Enzymatic Adenylation of mRNA

Two mRNAs were enzymatically adenylated:

-   -   ppLuc(GC)-ag-A64 and ppLuc(GC)-ag-histoneSL.

To this end, RNA was incubated with E. coli Poly(A)-polymerase and ATP(Poly(A) Polymerase Tailing Kit, Epicentre, Madison, USA) following themanufacturer's instructions. mRNA with extended poly(A) sequence waspurified and resuspended in water. The length of the poly(A) sequencewas determined via agarose gel electrophoresis. Starting mRNAs wereextended by approximately 250 adenylates, the mRNAs obtained aredesignated as ppLuc(GC)-ag-A300 and ppLuc(GC)-ag-histoneSL-A250,respectively.

5. Luciferase Expression by mRNA Electroporation

HeLa cells were trypsinized and washed in opti-MEM. 1×10⁵ cells in 200μl of opti-MEM each were electroporated with 0.5 μg of ppLuc-encodingmRNA. As a control, mRNA not coding for ppLuc was electroporatedseparately. Electroporated cells were seeded in 24-well plates in 1 mlof RPMI 1640 medium. 6, 24, or 48 hours after transfection, medium wasaspirated and cells were lysed in 200 μl of lysis buffer (25 mM Tris, pH7.5 (HCl), 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM DTT, 1 mMPMSF). Lysates were stored at −20° C. until ppLuc activity was measured.

6. Luciferase Expression by mRNA Lipofection

HeLa cells were seeded in 96 well plates at a density of 2×10⁴ cells perwell. The following day, cells were washed in opti-MEM and thentransfected with 0.25 μg of Lipofectin-complexed ppLuc-encoding mRNA in150 μl of opti-MEM. As a control, mRNA not coding for ppLuc waslipofected separately. In some wells, opti-MEM was aspirated and cellswere lysed in 200 μl of lysis buffer 6 hours after the start oftransfection. In the remaining wells, opti-MEM was exchanged for RPMI1640 medium at that time. In these wells, medium was aspirated and cellswere lysed in 200 μl of lysis buffer 24 or 48 hours after the start oftransfection. Lysates were stored at −20° C. until ppLuc activity wasmeasured.

7. Luciferase Measurement

-   -   ppLuc activity was measured as relative light units (RLU) in a        BioTek SynergyHT plate reader at 5 seconds measuring time using        50 μl of lysate and 200 μl of luciferin buffer (25 mM        Glycylglycin, pH 7.8 (NaOH), 15 mM MgSO₄, 2 mM ATP, 75 μM        luciferin). Specific RLU were calculated by subtracting RLU of        the control RNA from total RLU.

8. Luciferase Expression by Intradermal mRNA Injection (LuciferaseExpression In Vivo)

Mice were anaesthetized with a mixture of Rompun and Ketavet. EachppLuc-encoding mRNA was injected intradermally (0.5 μg of mRNA in 50 μlper injection). As a control, mRNA not coding for ppLuc was injectedseparately. 16 hours after injection, mice were sacrificed and tissuecollected. Tissue samples were flash frozen in liquid nitrogen and lysedin a tissue lyser (Qiagen) in 800 μl of lysis buffer (25 mM Tris, pH 7.5(HCl), 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM DTT, 1 mM PMSF).Subsequently samples were centrifuged at 13500 rpm at 4° C. for 10minutes. Lysates were stored at −80° C. until ppLuc activity wasmeasured (see 7. luciferase measurement).

9. NY-ESO-1 Expression by mRNA Electroporation

HeLa cells were trypsinized and washed in opti-MEM. 2×10⁵ cells in 200μl of opti-MEM were electroporated with 10 μg of NY-ESO-1-encoding mRNA.Cells from three electroporations were combined and seeded in a 6-wellplate in 2 ml of RPMI 1640 medium. 24 hours after transfection, cellswere harvested and transferred into a 96 well V-bottom plate (2 wellsper mRNA). Cells were washed with phosphate buffered saline (PBS) andpermeabilized with 200 μl per well of Cytofix/Cytoperm (Becton Dickinson(BD)). After 15 minutes, cells were washed with PERM/WASH® buffer (BD).Then, cells were incubated for 1 hour at room temperature with eithermouse anti-NY-ESO-1 IgG1 or an isotype control (20 μg/ml). Cells werewashed twice with PERM/WASH® buffer again. Next, cells were incubatedfor 1 hour at 4° C. with a 1:500 dilution of Alexa-647 coupledgoat-anti-mouse IgG. Finally, cells were washed twice with PERM/WASH®buffer. Cells were resuspended in 200 μl of buffer (PBS, 2% FCS, 2 mMEDTA, 0.01% sodium azide). NY-ESO-1 expression was quantified by flowcytometry as median fluorescence intensity (MFI).

10. Induction of Anti NY-ESO-1 Antibodies by Vaccination with mRNA

C57BL/6 mice were vaccinated intradermally with NY-ESO-1-encoding mRNAcomplexed with protamine (5 times in 14 days). Control mice were treatedwith buffer. The level of NY-ESO-1-specific antibodies in vaccinated andcontrol mice was analyzed 8 days after the last vaccination by ELISA: 96well ELISA plates (Nunc) were coated with 100 μl per well of 10 μg/mlrecombinant NY-ESO-1 protein for 16 hours at 4° C. Plates were washedtwo times with wash buffer (PBS, 0.05% TWEEN® 20 non-ionic detergent).To block unspecific binding, plates were then incubated for 2 hours at37° C. with blocking buffer (PBS, 0.05% TWEEN® 20 non-ionic detergent,1% BSA). After blocking, 100 μl per well of serially diluted mouse serawere added and incubated for 4 hours at room temperature. Plates werethen washed three times with wash buffer. Next, 100 μl per well ofbiotinylated rat anti-mouse IgG2a[b] detection antibody (BD Biosciences)diluted 1:600 in blocking buffer was allowed to bind for 1 hour at roomtemperature. Plates were washed again three times with wash buffer,followed by incubation for 30 minutes at room temperature with 100 μlper well of horseradish peroxidase-coupled streptavidin. After fourwashes with wash buffer, 100 μl per well of3,3′,5,5′-tetramethylbenzidine (Thermo Scientific) was added. Upon theresulting change in color 100 μl per well of 20% sulfuric acid wasadded. Absorbance was measured at 405 nm.

11. Induction of Anti Survivin Antibodies by Vaccination with mRNA

C57BL/6 mice were vaccinated intradermally with Survivin-encoding mRNAcomplexed with protamine (5 times in 14 days). Control mice were treatedwith buffer. The level of Survivin-specific antibodies in vaccinated andcontrol mice was analyzed 8 days after the last vaccination by ELISA: 96well ELISA plates (Nunc) were coated with 100 μl per well of 10 μg/mlrecombinant Survivin protein for 16 hours at 4° C. Plates were washedtwo times with wash buffer (PBS, 0.05% TWEEN® 20 non-ionic detergent).To block unspecific binding, plates were then incubated for 2 hours at37° C. with blocking buffer (PBS, 0.05% TWEEN® 20 non-ionic detergent,1% BSA). After blocking, 100 μl per well of serially diluted mouse serawere added and incubated for 4 hours at room temperature. Plates werethen washed three times with wash buffer. Next, 100 μl per well ofbiotinylated rat anti-mouse IgG2a[b] detection antibody (BD Biosciences)diluted 1:600 in blocking buffer was allowed to bind for 1 hour at roomtemperature. Plates were washed again three times with wash buffer,followed by incubation for 30 minutes at room temperature with 100 μlper well of horseradish peroxidase-coupled streptavidin. After fourwashes with wash buffer, 100 μl per well of3,3′,5,5′-tetramethylbenzidine (Thermo Scientific) was added. Upon theresulting change in color 100 μl per well of 20% sulfuric acid wasadded. Absorbance was measured at 405 nm.

12. Induction of Anti MAGE-C1 Antibodies by Vaccination with mRNA

C57BL/6 mice were vaccinated intradermally with MAGE-C1-encoding mRNAcomplexed with protamine (5 times in 14 days). Control mice were treatedwith buffer. The level of MAGE-C1-specific antibodies in vaccinated andcontrol mice was analyzed 8 days after the last vaccination by ELISA: 96well ELISA plates (Nunc) were coated with 100 μl per well of 10 μg/mlrecombinant MAGE-C1 protein for 16 hours at 4° C. Plates were washed twotimes with wash buffer (PBS, 0.05% TWEEN® 20 non-ionic detergent). Toblock unspecific binding, plates were then incubated for 2 hours at 37°C. with blocking buffer (PBS, 0.05% TWEEN® 20 non-ionic detergent, 1%BSA). After blocking, 100 μl per well of serially diluted mouse serawere added and incubated for 4 hours at room temperature. Plates werethen washed three times with wash buffer. Next, 100 μl per well ofbiotinylated rat anti-mouse IgG2a[b] detection antibody (BD Biosciences)diluted 1:600 in blocking buffer was allowed to bind for 1 hour at roomtemperature. Plates were washed again three times with wash buffer,followed by incubation for 30 minutes at room temperature with 100 μlper well of horseradish peroxidase-coupled streptavidin. After fourwashes with wash buffer, 100 μl per well of3,3′,5,5′-tetramethylbenzidine (Thermo Scientific) was added. Upon theresulting change in color 100 μl per well of 20% sulfuric acid wasadded. Absorbance was measured at 405 nm.

13. Detection of an Antigen-Specific Cellular Immune Response (T CellImmune Response) by ELISPOT:

C57BL/6 mice are vaccinated intradermally with MAGE-C1 encoding mRNAcomplexed with protamine (5 times in 14 days). Control mice are treatedwith buffer. 1 week after the last vaccination mice are sacrificed, thespleens are removed and the splenocytes are isolated. The splenocytesare restimulated for 7 days in the presence of peptides from the aboveantigen (peptide library) or coincubated with dendritic cells generatedfrom bone marrow cells of native syngeneic mice, which areelectroporated with mRNA coding for the antigen. To determine anantigen-specific cellular immune response INFgamma secretion wasmeasured after re-stimulation. For detection of INFgamma a coatmultiscreen plate (Millipore) is incubated overnight with coating buffer0.1 M carbonate-bicarbonate buffer pH 9.6, 10.59 g/l Na₂CO₃, 8.4 g/lNaHCO₃) comprising antibody against INFγ (BD Pharmingen, Heidelberg,Germany). Stimulators and effector cells are incubated together in theplate in the ratio of 1:20 for 24 h. The plate is washed with 1×PBS andincubated with a biotin-coupled secondary antibody. After washing with1×PBS/0.05% TWEEN® 20 non-ionic detergent, the substrate(5-Bromo-4-Cloro-3-Indolyl Phosphate/Nitro Blue Tetrazolium LiquidSubstrate System from Sigma Aldrich, Taufkirchen, Germany) is added tothe plate and the conversion of the substrate could be detectedvisually.

14. Results

14.1 Histone Stem-Loop Sequences:

In order to characterize histone stem-loop sequences, sequences frommetazoa and protozoa (4001 sequences), or from protozoa (131 sequences)or alternatively from metazoa (3870 sequences), or from vertebrates(1333 sequences) or from humans (84 sequences) were grouped and aligned.Then, the quantity of the occurring nucleotides was determined for everyposition. Based on the tables thus obtained, consensus sequences for the5 different groups of sequences were generated representing allnucleotides present in the sequences analyzed. Within the consensussequence of metazoa and protozoa combined, 3 nucleotides are conserved,a T/U in the loop and a G and a C in the stem, forming a base pair.Structurally, typically a 6 base-pair stem and a loop of 4 nucleotidesis formed. However, deviating structures are common: Of 84 human histonestem-loops, two contain a stem of only 5 nucleotides comprising 4base-pairs and one mismatch. Another human histone stem-loop contains astem of only 5 base-pairs. Four more human histone stem-loops contain a6 nucleotide long stem, but include one mismatch at three differentpositions, respectively. Furthermore, four human histone stem-loopscontain one wobble base-pair at two different positions, respectively.Concerning the loop, a length of 4 nucleotides seems not to be strictlyrequired, as a loop of 5 nucleotides has been identified in D.discoideum.

In addition to the consensus sequences representing all nucleotidespresent in the sequences analyzed, more restrictive consensus sequenceswere also obtained, increasingly emphasizing conserved nucleotides. Insummary, the following sequences were obtained:

-   -   (Cons): represents all nucleotides present    -   (99%): represents at least 99% of all nucleotides present    -   (95%): represents at least 95% of all nucleotides present    -   (90%): represents at least 90% of all nucleotides present

The results of the analysis of histone stem-loop sequences aresummarized in the following Tables 1 to 5 (see also FIG. 1 to 5):

TABLE 1 Metazoan and protozoan histone stem-loop consensus sequence:(based on an alignment of 4001 metazoan and protozoan histone stem-loopsequences) (see also FIG. 1) < < < < < < · · # A 2224 1586 3075 28721284 184 0 13 12 9 1 47 59 # T 172 188 47 205 19 6 0 569 1620 199 39473830 3704 # C 1557 2211 875 918 2675 270 0 3394 2342 3783 51 119 227 # G25 16 4 6 23 3541 4001 25 27 10 2 5 11 Cons N* N* N N N N G N N N N N N99% H* H* H H V V G Y Y Y Y H H 95% M* H* M H M S G Y Y Y T T Y 90% M*M* M M M S G Y Y C T T T · · > > > > > > # A 0 675 3818 195 1596 523 014 3727 61 771 2012 2499 # T 4001 182 1 21 15 11 0 179 8 64 557 201 690# C 0 3140 7 50 31 16 4001 3543 154 3870 2636 1744 674 # G 0 4 175 37352359 3451 0 265 112 4 37 43 138 Cons T N N N N N C N N N N* N* N* 99% TH R V V R C B V H H* N* N* 95% T M A R R R C S M C H* H* H* 90% T M A GR R C S A C H* M* H*

TABLE 2 Protozoan histone stem-loop consensus sequence: (based on analignment of 131 protozoan histone stem-loop sequences) (see also FIG.2) < < < < < < · · · · > > > > > > # A 52 32 71 82 76 13 0 12 12  9 1 463 0 75 82 53 79 20 0 4 94 17 35 74 56 # T 20 32 37 21 8 3 0 21 85 58 8670 65 131  28 1 17 13 10 0 15 7 31 32 20 28 # C 45 59 20 25 38 0 0 86  854 42 13 58 0 27 2  6 31 10 131  112 5 82 58 30 40 # G 14  8  3  3 9 115131 12 26 10 2  2 5 0  1 46 55  8 91 0 0 25  1  6  7  7 Cons N* N* N N ND G N N N N N N T N N N N N C H N N N* N* N* 99% N* N* N N N D G N N N BN N T H V N N N C H N H N* N* N* 95% N* N* H H N R G N N N Y H B T H R DN N C Y D H H* N* N* 90% N* H* H H V R G N D B Y H Y T H R D H N C Y R HH* H* H*

TABLE 3 Metazoan histone stem-loop consensus sequence: (based on analignment of 3870 (including 1333 vertebrate sequences) metazoan histonestem-loop sequences) (see also FIG. 3) < < < < < < · · # A 2172 15543004 2790 1208 171 0 1 0 0 0 1 56 # T 152 156 10 184 11 3 0 548 1535 1413861 3760 3639 # C 1512 2152 855 893 2637 270 0 3308 2334 3729 9 106 169# G 11 8 1 3 14 3426 3870 13 1 0 0 3 6 Cons N* N* N N N N G N B Y Y N N99% H* H* M H M V G Y Y Y T Y H 95% M* M* M M M S G Y Y C T T Y 90% M*M* M M M S G Y Y C T T T · · > > > > > > # A 0 600 3736 142 1517 503 010 3633 44 736 1938 2443 # T 3870 154 0 4 2 1 0 164 1 33 525 181 662 # C0 3113 5 44 0 6 3870 3431 149 3788 2578 1714 634 # G 0 3 129 3680 23513360 0 265 87 3 31 36 131 Cons T N V N D N C N N N N* N* N* 99% T H R VR R C B V M H* H* N* 95% T M A G R R C S M C H* H* H* 90% T M A G R R CS A C H* M* H*

TABLE 4 Vertebrate histone stem-loop consensus sequence: (based on analignment of 1333 vertebrate histone stem-loop sequences) (see also FIG.4) < < < < < < · · # A 661 146 1315 1323 920 8 0 1 0 0 0 1 4 # T 63 1212 2 6 2 0 39 1217 2 1331 1329 1207 # C 601 1062 16 6 403 1 0 1293 1161331 2 0 121 # G 8 4 0 2 4 1322 1333 0 0 0 0 3 1 Cons N* N* H N N N G HY Y Y D N 99% H* H* M A M G G Y Y C T T Y 95% H* H* A A M G G C Y C T TY 90% M* M* A A M G G C T C T T T · · > > > > > > # A 0 441 1333 0 119921 0 1 1126 26 81 380 960 # T 1333 30 0 1 0 1 0 2 1 22 91 91 12 # C 0862 0 2 0 0 1333 1328 128 1284 1143 834 361 # G 0 0 0 1330 134 1311 0 278 1 18 28 0 Cons T H A B R D C N N N N* N* H* 99% T H A G R R C C V HN* N* M* 95% T M A G R G C C V C H* H* M* 90% T M A G R G C C M C Y* M*M*

TABLE 5 Homo sapiens histone stem-loop consensus sequence: (based on analignment of 84 human histone stem-loop sequences) (see also FIG. 5) < << < < < · · · · > > > > > > # A 10 17 84 84 76 1 0 1 0 0 0 1 0 0 12 84 065 3 0 0 69 5 0 10 64 # T 8 6 0 0 2 2 0 1 67 0 84 80 81 84 5 0 0 0 0 0 00 4 25 24 3 # C 62 61 0 0 6 0 0 82 17 84 0 0 3 0 67 0 1 0 0 84 84 5 7557 44 17 # G 4 0 0 0 0 81 84 0 0 0 0 3 0 0 0 0 83 19 81 0 0 10 0 2 6 0Cons N* H* A A H D G H Y C T D Y T H A S R R C C V H B* N* H* 99% N* H*A A H D G H Y C T D Y T H A S R R C C V H B* N* H* 95% H* H* A A M G G CY C T T T T H A G R G C C V M Y* N* M* 90% H* M* A A A G G C Y C T T T TM A G R G C C R M Y* H* M*

Wherein the used abbreviations were defined as followed:

abbreviation Nucleotide bases remark G G Guanine A A Adenine T T ThymineU U Uracile C C Cytosine R G or A Purine Y T/U or C Pyrimidine M A or CAmino K G or T/U Keto S G or C Strong (3H bonds) W A or T/U Weak (2Hbonds) H A or C or T/U Not G B G or T/U or C Not A V G or C or A Not T/UD G or A or T/U Not C N* G or C or T/U or A Any base present or not Basemay be present or not

14.2 the Combination of Poly(A) and histoneSL Increases ProteinExpression from mRNA in a Synergistic Manner.

To investigate the effect of the combination of poly(A) and histoneSL onprotein expression from mRNA, mRNAs with different sequences 3′ of thealpha-globin 3′-UTR were synthesized: mRNAs either ended just 3′ of the3′-UTR, thus lacking both poly(A) sequence and histoneSL, or containedeither an A64 poly(A) sequence or a histoneSL instead, or both A64poly(A) and histoneSL 3′ of the 3′-UTR. Luciferase-encoding mRNAs orcontrol mRNA were electroporated into HeLa cells. Luciferase levels weremeasured at 6, 24, and 48 hours after transfection (see following Table6 and FIG. 22).

TABLE 6 RLU at RLU at RLU at mRNA 6 hours 24 hours 48 hoursppLuc(GC)-ag-A64-histoneSL 466553 375169 70735 ppLuc(GC)-ag-histoneSL50947 3022 84 ppLuc(GC)-ag-A64 10471 19529 4364 ppLuc(GC)-ag 997 217 42

Little luciferase was expressed from mRNA having neither poly(A)sequence nor histoneSL. Both a poly(A) sequence or the histoneSLincreased the luciferase level to a similar extent. Either mRNA gaverise to a luciferase level much higher than did mRNA lacking bothpoly(A) and histoneSL. Strikingly however, the combination of poly(A)and histoneSL further strongly increased the luciferase level, manifoldabove the level observed with either of the individual elements. Themagnitude of the rise in luciferase level due to combining poly(A) andhistoneSL in the same mRNA demonstrates that they are actingsynergistically.

The synergy between poly(A) and histoneSL was quantified by dividing thesignal from poly(A)-histoneSL mRNA (+/+) by the sum of the signals fromhistoneSL mRNA (−/+) plus poly(A) mRNA (+/−) (see following Table 7).

TABLE 7 RLU at RLU at RLU at A64 histoneSL 6 hours 24 hours 48 hours + +466553 375169 70735 − + 50947 3022 84 + − 10471 19529 4364 Synergy 7.616.6 15.9

The factor thus calculated specifies how much higher the luciferaselevel from mRNA combining poly(A) and histoneSL is than would beexpected if the effects of poly(A) and histoneSL were purely additive.The luciferase level from mRNA combining poly(A) and histoneSL was up to16.6 times higher than if their effects were purely additive. Thisresult confirms that the combination of poly(A) and histoneSL effects amarkedly synergistic increase in protein expression.

14.3 the Combination of Poly(A) and histoneSL Increases ProteinExpression from mRNA Irrespective of their Order.

The effect of the combination of poly(A) and histoneSL might depend onthe length of the poly(A) sequence and the order of poly(A) andhistoneSL. Thus, mRNAs with increasing poly(A) sequence length and mRNAwith poly(A) and histoneSL in reversed order were synthesized: Two mRNAscontained 3′ of the 3′-UTR either an A120 or an A300 poly(A) sequence.One further mRNA contained 3′ of the 3′-UTR first a histoneSL followedby an A250 poly(A) sequence. Luciferase-encoding mRNAs or control mRNAwere lipofected into HeLa cells. Luciferase levels were measured at 6,24, and 48 hours after the start of transfection (see following Table 8and FIG. 23).

TABLE 8 RLU at RLU at RLU at mRNA 6 hours 24 hours 48 hoursppLuc(GC)-ag-histoneSL-A250 98472 734222 146479ppLuc(GC)-ag-A64-histoneSL 123674 317343 89579 ppLuc(GC)-ag-histoneSL7291 4565 916 ppLuc(GC)-ag-A300 4357 38560 11829 ppLuc(GC)-ag-A120 437145929 10142 ppLuc(GC)-ag-A64 1928 26781 537

Both an A64 poly(A) sequence or the histoneSL gave rise to comparableluciferase levels. In agreement with the previous experiment did thecombination of A64 and histoneSL strongly increase the luciferase level,manifold above the level observed with either of the individualelements. The magnitude of the rise in luciferase level due to combiningpoly(A) and histoneSL in the same mRNA demonstrates that they are actingsynergistically. The synergy between A64 and histoneSL was quantified asbefore based on the luciferase levels of A64-histoneSL, A64, andhistoneSL mRNA (see following Table 9). The luciferase level from mRNAcombining A64 and histoneSL was up to 61.7 times higher than if theeffects of poly(A) and histoneSL were purely additive.

TABLE 9 RLU at RLU at RLU at A64 histoneSL 6 hours 24 hours 48 hours + +123674 317343 89579 − + 7291 4565 916 + − 1928 26781 537 Synergy 13.410.1 61.7

In contrast, increasing the length of the poly(A) sequence from A64 toA120 or to A300 increased the luciferase level only moderately (seeTable 8 and FIG. 19). mRNA with the longest poly(A) sequence, A300, wasalso compared to mRNA in which a poly(A) sequence of similar length wascombined with the histoneSL, histoneSL-A250. In addition to having along poly(A) sequence, the order of histoneSL and poly(A) is reversed inthis mRNA relative to A64-histoneSL mRNA. The combination of A250 andhistoneSL strongly increased the luciferase level, manifold above thelevel observed with either histoneSL or A300. Again, the synergy betweenA250 and histoneSL was quantified as before comparing RLU fromhistoneSL-A250 mRNA to RLU from A300 mRNA plus histoneSL mRNA (seefollowing Table 10). The luciferase level from mRNA combining A250 andhistoneSL was up to 17.0 times higher than if the effects of poly(A) andhistoneSL were purely additive.

TABLE 10 RLU at RLU at RLU at histoneSL A250/A300 6 hours 24 hours 48hours + + 98472 734222 146479 + − 7291 4565 916 − + 4357 38560 11829Synergy 8.5 17.0 11.5

In summary, a highly synergistic effect of the combination of histoneSLand poly(A) on protein expression from mRNA has been demonstrated forsubstantially different lengths of poly(A) and irrespective of the orderof poly(A) and histoneSL.

14.4 the Rise in Protein Expression by the Combination of Poly(A) andhistoneSL is Specific

To investigate whether the effect of the combination of poly(A) andhistoneSL on protein expression from mRNA is specific, mRNAs withalternative sequences in combination with poly(A) were synthesized:These mRNAs contained 3′ of A64 one of seven distinct sequences,respectively. Luciferase-encoding mRNAs or control mRNA wereelectroporated into HeLa cells. Luciferase levels were measured at 6,24, and 48 hours after transfection (see following Table 11 and FIG.24).

TABLE 11 RLU at RLU at RLU at mRNA 6 hours 24 hours 48 hoursppLuc(GC)-ag-A64-N32 33501 38979 2641 ppLuc(GC)-ag-A64-SL 28176 20364874 ppLuc(GC)-ag-A64-U30 41632 54676 3408 ppLuc(GC)-ag-A64-G30 4676349210 3382 ppLuc(GC)-ag-A64-PolioCL 46428 26090 1655ppLuc(GC)-ag-A64-aCPSL 34176 53090 3338 ppLuc(GC)-ag-A64-ag 18534 18194989 ppLuc(GC)-ag-A64-histoneSL 282677 437543 69292ppLuc(GC)-ag-histoneSL 27597 3171 0 ppLuc(GC)-ag-A64 14339 48414 9357

Both a poly(A) sequence or the histoneSL gave rise to comparableluciferase levels. Again, the combination of poly(A) and histoneSLstrongly increased the luciferase level, manifold above the levelobserved with either of the individual elements, thus actingsynergistically. In contrast, combining poly(A) with any of thealternative sequences was without effect on the luciferase levelcompared to mRNA containing only a poly(A) sequence. Thus, thecombination of poly(A) and histoneSL increases protein expression frommRNA in a synergistic manner, and this effect is specific.

14.5 the Combination of Poly(A) and histoneSL Increases ProteinExpression from mRNA in a Synergistic Manner In Vivo.

To investigate the effect of the combination of poly(A) and histoneSL onprotein expression from mRNA in vivo, Luciferase-encoding mRNAs withdifferent sequences 3′ of the alpha-globin 3′-UTR or control mRNA wereinjected intradermally into mice: mRNAs contained either an A64 poly(A)sequence or a histoneSL instead, or both A64 poly(A) and histoneSL 3′ ofthe 3′-UTR. Luciferase levels were measured at 16 hours after injection(see following Table 12 and FIG. 25).

TABLE 12 RLU at mRNA 16 hours ppLuc(GC)-ag-A64-histoneSL 38081ppLuc(GC)-ag-histoneSL 137 ppLuc(GC)-ag-A64 4607

Luciferase was expressed from mRNA having either a histoneSL or apoly(A) sequence. Strikingly however, the combination of poly(A) andhistoneSL further strongly increased the luciferase level, manifoldabove the level observed with either of the individual elements. Themagnitude of the rise in luciferase level due to combining poly(A) andhistoneSL in the same mRNA demonstrates that they are actingsynergistically.

The synergy between poly(A) and histoneSL was quantified by dividing thesignal from poly(A)-histoneSL mRNA (+/+) by the sum of the signals fromhistoneSL mRNA (−/+) plus poly(A) mRNA (+/−) (see following Table 13).

TABLE 13 RLU at A64 histoneSL 16 hours + + 38081 − + 137 + − 4607Synergy 8.0

The factor thus calculated specifies how much higher the luciferaselevel from mRNA combining poly(A) and histoneSL is than would beexpected if the effects of poly(A) and histoneSL were purely additive.The luciferase level from mRNA combining poly(A) and histoneSL was 8times higher than if their effects were purely additive. This resultconfirms that the combination of poly(A) and histoneSL effects amarkedly synergistic increase in protein expression in vivo.

14.6 the Combination of Poly(A) and histoneSL Increases NY-ESO-1 ProteinExpression from mRNA.

To investigate the effect of the combination of poly(A) and histoneSL onprotein expression from mRNA, NY-ESO-1-encoding mRNAs with differentsequences 3′ of the alpha-globin 3′-UTR were synthesized: mRNAscontained either an A64 poly(A) sequence or both A64 poly(A) andhistoneSL 3′ of the 3′-UTR. NY-ESO-1-encoding mRNAs were electroporatedinto HeLa cells. NY-ESO-1 levels were measured at 24 hours aftertransfection by flow cytometry (see following Table 14 and FIG. 26).

TABLE 14 MFI at 24 hours mRNA anti-NY-ESO-1 isotype controlNY-ESO-1(GC)-ag-A64-histoneSL 15600 1831 NY-ESO-1(GC)-ag-A64 1294 849

NY-ESO-1 was expressed from mRNA having only a poly(A) sequence.Strikingly however, the combination of poly(A) and histoneSL stronglyincreased the NY-ESO-1 level, manifold above the level observed withonly a poly(A) sequence.

14.7 the Combination of Poly(A) and histoneSL Increases the Level ofAntibodies Elicited by Vaccination with mRNA.

To investigate the effect of the combination of poly(A) and histoneSL onthe induction of antibodies elicited by vaccination with mRNA, C57BL/6mice were vaccinated intradermally with protamine-complexed,NY-ESO-1-encoding mRNAs with different sequences 3′ of the alpha-globin3′-UTR. mRNAs contained either an A64 poly(A) sequence or both A64poly(A) and histoneSL 3′ of the 3′-UTR. The level of NY-ESO-1-specificantibodies in vaccinated and control mice was analyzed by ELISA withserial dilutions of sera (see following Table 15 and FIG. 27).

TABLE 15 mRNA mean IgG2a[b] endpoint titer NY-ESO-1(GC)-ag-A64-histoneSL763 NY-ESO-1(GC)-ag-A64 20

Anti NY-ESO-1 IgG2a[b] was induced by mRNA having only a poly(A)sequence. Strikingly however, the combination of poly(A) and histoneSLstrongly increased the anti NY-ESO-1 IgG2a[b] level, manifold above thelevel observed with only a poly(A) sequence.

The invention claimed is:
 1. A nucleic acid molecule comprising: (I) aDNA molecule coding for, from 5 to 3′: a) a polypeptide coding region,encoding a tumour antigen or an epitope of a tumour antigen; b) apoly(A) sequence or a polyadenylation signal, and c) at least onehistone stem-loop that encodes a RNA that specifically binds tostem-loop binding protein (SLBP) without a histone downstream element(HDE); or (II) a RNA molecule comprising, from 5′ to 3′: a) apolypeptide coding region, encoding a tumour antigen or an epitope of atumour antigen; b) a poly(A) sequence, and c) at least one histonestem-loop that specifically binds to SLBP without a HDE.
 2. The nucleicacid molecule according to claim 1, wherein the tumour antigen or theepitope of a tumour antigen is selected from 5T4, 707-AP, 9D7, AFP,AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin,alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m,B7H4, B AGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m,CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m,cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4,CD52, CD55, CD56, CD80, CDCl27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28,CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6,Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN,EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1,EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2,hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE,homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M,HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature lamininreceptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m,KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3,MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1,MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1,MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrixprotein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CAIX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin classI/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m,NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1,OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE,PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP-1,survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP,TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1,TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2/FLK-1, andor WT1.
 3. The nucleic acid molecule of claim 1, wherein the moleculedoes not comprise a sequence encoding a reporter protein, a marker, or aselection protein.
 4. The nucleic acid molecule of claim 1, wherein thenucleic acid is an RNA.
 5. The nucleic acid molecule of claim 1, whereinthe poly(A) sequence comprises a sequence of about 25 to about 400adenosine nucleotides.
 6. The nucleic acid molecule of claim 1, whereinthe polyadenylation signal comprises the consensus sequence NN(U/T)ANA.7. The nucleic acid molecule of claim 1, wherein at least one guanosine,uridine, adenosine, thymidine, or cytidine position of the nucleic acidmolecule is substituted with an analogue of these nucleotides selectedfrom 2-amino-6-chloropurineriboside-5′-triphosphate,2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-iodouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate,5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate,6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, 06-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, andxanthosine-5′-triphosphate.
 8. The nucleic acid sequence molecule ofclaim 1, wherein the G/C content of the polypeptide coding region isincreased compared with the G/C content of the coding region of awild-type nucleic acid encoding the tumour antigen.
 9. The nucleic acidmolecule of claim 4, wherein the RNA comprises a 5′ cap structure and apoly(A) sequence of about 25 to about 400 adenosine nucleotides.
 10. Thenucleic acid molecule of claim 1, wherein the nucleic acid moleculecomprises a sequence of at least 10 consecutive cytidines.
 11. Thenucleic acid molecule of claim 1, wherein the nucleic acid moleculefurther comprises a stabilizing sequence from the alpha globin 3′ UTR,positioned 3′ relative to the polypeptide coding region of the nucleicacid molecule.
 12. A pharmaceutical composition comprising a nucleicacid molecule of claim 1 and a pharmaceutically acceptable carrier. 13.The pharmaceutical composition of claim 12, further comprising anadjuvant.
 14. The pharmaceutical composition of claim 12, wherein thecomposition further comprises a cationic or polycationic compound incomplex with the nucleic acid molecule.
 15. The pharmaceuticalcomposition of claim 12, wherein the composition further comprises apolycationic polypeptide in complex with the nucleic acid molecule. 16.The nucleic acid molecule of claim 4, wherein RNA comprises apolypeptide coding region, encoding an epitope of a tumour antigen. 17.The nucleic acid molecule of claim 16, wherein the epitope of a tumourantigen is a T-cell epitope.
 18. The nucleic acid molecule of claim 16,wherein the epitope is at least one epitope from KRAS.